Pegylated c-peptide

ABSTRACT

The present invention relates to modified forms of C-peptide, and methods for their use. In one aspect, the modified forms of C-peptide comprise PEGylated C-peptide derivatives comprising at least one PEG group attached to the N-terminus, which exhibit superior pharmacokinetic and biological activity in vivo.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Application No. 13/109,522, filedMay 17, 2011, which claims the benefit of priority of U.S. provisionalapplications Nos. 61/345,293, filed May 17, 2010 and 61/448,402, filedMar. 2, 2011, the disclosures of which are incorporated by reference asif written herein in their entireties.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to modified forms of C-peptide, andmethods for their use. In one aspect, the modified forms of C-peptidecomprise PEGylated C-peptide derivatives comprising at least one PEGgroup attached to the N-terminus which exhibit superior pharmacokineticand biological activity in vivo.

C-peptide is the linking peptide between the A- and B-chains in theproinsulin molecule. After cleavage and processing in the endoplasmicreticulum of pancreatic islet β-cells, insulin and C-peptide aregenerated. C-peptide is co-secreted with insulin in equimolar amountsfrom the pancreatic islet β-cells into the portal circulation. Besidesits contribution to the folding of the two-chain insulin structure,further biologic activity of C-peptide was questioned for many yearsafter its discovery.

Type 1 diabetes, or insulin-dependent diabetes mellitus, is generallycharacterized by insulin and C-peptide deficiency, due to an autoimmunedestruction of the pancreatic islet β-cells. The patients are thereforedependent on exogenous insulin to sustain life. Several factors may beof importance for the pathogenesis of the disease, e.g., geneticbackground, environmental factors, and an aggressive autoimmune reactionfollowing a temporary infection (Akerblom H K et al.: Annual Medicine29(5): 383-385, (1997)). Currently insulin-dependent diabetics areprovided with exogenous insulin which has been separated from theC-peptide, and thus do not receive exogenous C-peptide therapy. Bycontrast most type 2 diabetics initially still produce both insulin andC-peptide endogenously, but are generally characterized by insulinresistance in skeletal muscle and adipose tissue.

Type 1 diabetics suffer from a constellation of long-term complicationsof diabetes that are in many cases more severe and widespread than intype 2 diabetes. Specifically, for example microvascular complicationsinvolving the retina, kidneys, and nerves are a major cause of morbidityand mortality in patients with type 1 diabetes.

There is increasing support for the concept that C-peptide deficiencymay play a role in the development of the long-term complications ofinsulin-dependent diabetics. Additionally, in vivo as well as in vitrostudies, in diabetic animal models and in patients with type 1 diabetes,demonstrate that C-peptide possesses hormonal activity (Wahren J et al.:American Journal of Physiology 278: E759-E768, (2000); Wahren J et al.:In International textbook of diabetes mellitus Ferranninni E, Zimmet P,De Fronzo R A, Keen H, Eds. Chichester, John Wiley & Sons, (2004), p.165-182). Thus, C-peptide used as a complement to regular insulintherapy may provide an effective approach to the management of type 1diabetes long-term complications.

Studies to date suggest that C-peptide's therapeutic activity involvesthe binding of C-peptide to a G-protein-coupled membrane receptor,activation of Ca²⁺-dependent intracellular signalling pathways, andphosphorylation of the MAP-kinase system, eliciting increased activitiesof sodium/potassium ATPase and endothelial nitric oxide synthase (eNOS).Despite the promise of using C-peptide to treat and prevent thelong-term complications of insulin-dependent diabetes, the shortbiological half-life and requirement to dose C-peptide multiple timesper day via subcutaneous injection, or intravenous (I.V.)administration, has hindered commercial development.

The present invention is focused on the development of PEGylatedversions of C-peptide that retain the biological activity of the nativeC-peptide and exhibit superior pharmacokinetic properties. Theseimproved therapeutic forms of C-peptide enable the development of moreeffective therapeutic regimens for the treatment of the long-termcomplications of diabetes, and require significantly less frequentadministration.

In one aspect, these therapies are targeted to diabetic patients, and ina further aspect to insulin-dependent patients. In one aspect, theinsulin-dependent patients are suffering from one or more long-termcomplications of diabetes.

These improved methods are based on animal studies that surprisinglydemonstrate that modification of C-peptide at the N-terminus of themolecule results in PEGylated versions of C-peptide that retain thebiological activity of the native molecule, while exhibiting vastlysuperior pharmacokinetic characteristics.

SUMMARY OF THE INVENTION

In one embodiment, the present invention includes a PEGylated C-peptidecomprising a PEG moiety covalently attached to the N-terminus ofC-peptide. In one aspect, the PEGylated C-peptide of the inventioncomprises a linear polymer PEG polymer. In another aspect, the PEGylatedC-peptide of the invention comprises a branched chain PEG polymer.

In another aspect of any of these PEGylated C-peptides, the PEG moietyhas a molecular weight of between about 10 kDa and about 80 kDa. Inanother aspect, the PEG moiety has a molecular weight of between about20 kDa and about 60 kDa. In another aspect, the PEG moiety has amolecular weight of between about 30 kDa and about 50 kDa.

In another aspect of any of these linear PEGylated C-peptides, thePEGylated C-peptide has the general formula (I):

R₁—O—(CH₂CH₂O)_(n1)-[Linker]-[C-peptide]  (I)

wherein;

-   -   R₁=alkyl;    -   n₁ is 20 to 800;    -   the linker selected from; —X—, —CO—, —(CH₂)_(m2)—,        —(CH₂)_(m1)—CO—, —CO—(CH₂)_(m1)—,    -   —CO—X—CO—, —(CH₂)_(m1)—X—(CH₂)_(m1)—,        —(CH₂)_(m1)—CO—(CH₂)_(m1)—,    -   —X—CO—X—, —X—(CH₂)_(m1)—X—, —CO—(CH₂)_(m1)—CO—,    -   —X—CO—(CH₂)_(m1)—, —(CH₂)_(m1)—CO—X—, —X—(CH₂)_(m1)—CO—X—,    -   —X—CO—(CH₂)_(m1)X—, —X—CO—(CH₂)_(m1)—CO—X—(CH₂)_(m1)—X—CO—,    -   —X—(CH₂)_(m1)—X—CO—(CH₂)_(m2)—,    -   —X—(CH₂)_(m1)—CO—X—(CH₂)_(m2)—,    -   —X—(CH₂)_(m1)—X—CO—(CH₂)_(m2)—X—,    -   —X—(CH₂)_(m1)—X—CO—(CH₂)_(m2)—CO—,    -   —X—(CH₂)_(m1)—CO—X—(CH₂)_(m2)—X—, and    -   —X—(CH₂)_(m1)—CO—X—(CH₂)_(m2)—CO—;

wherein;

-   -   each X is independently selected from —O—, —S—, or —NH— or is        missing; \    -   each m₁ is independently 0 to 5;    -   each m₂ is independently 1 to 5; and wherein the linker is        attached to the N-terminal amino group of C-peptide.

In another aspect of any of these linear PEGylated C-peptides, thePEGylated C-peptide comprises a linker connecting the PEG moiety toC-peptide selected from;

-   -   —X₁—(CH₂)_(m4)—CO—;    -   —X₁—CO—;    -   —X₁—CO—(CH₂)_(m4)—CO—;    -   —X₁—CO—X₂—(CH₂)_(m3)—CO—; and    -   X₁—(CH₂)_(m2)—X₂—CO—(CH₂)_(m4)—CO—;

wherein;

-   -   X₁ is —O—, or missing;    -   X₂ is —NH—;    -   m₂ is 1 to 5;    -   m₃ is 2; and    -   m₄ is 2 to 5.

In another embodiment, the present invention includes a PEGylatedC-peptide wherein the PEGylated C-peptide has the structure:

wherein n₁ is about 400 to about 1000.

In another embodiment, the present invention includes a PEGylatedC-peptide wherein the PEGylated C-peptide has the structure:

wherein n₁ is about 400 to about 1000.

In another embodiment, the present invention includes a PEGylatedC-peptide wherein the PEGylated C-peptide has the structure:

In another embodiment, the present invention includes a PEGylatedC-peptide wherein the PEGylated C-peptide has the structure:

wherein n is about 400 to about 1000.

In another aspect, the PEGylated C-peptide comprises a branched chainPEG of general formula: (II):

wherein;

-   -   R₁=alkyl;    -   n₁ is 20 to 800;    -   n₂ is 20 to 800; and wherein each linker is independently        defined as below, and, wherein the linker connecting to        C-peptide is attached to the N-terminal amino group of        C-peptide.

In another embodiment, the present invention includes a PEGylatedC-peptide wherein the PEGylated C-peptide has the structure of generalformula (IIA):

wherein;

-   -   R₁=alkyl;    -   n₁ is 20 to 800;    -   n₂ is 20 to 800;    -   the linker is selected from; —X—, —CO—, —(CH₂)_(m2)—,    -   —(CH₂)_(m1)—CO—, —CO—(CH₂)_(m1)—, —CO—X—CO—,        —(CH₂)_(m1)—X—(CH₂)_(m1)—,    -   —(CH₂)_(m1)—CO—(CH₂)_(m1)—, —X—CO—X—, —X—(CH₂)_(m1)—X—,        —CO—(CH₂)_(m1)—CO—,    -   —X—CO—(CH₂)_(m1)—, —(CH₂)_(m1)—CO—X—, —X—(CH₂)_(m1)—CO—X—,        —X—CO—(CH₂)_(m1)X—,    -   —X—CO—(CH₂)_(m1)—CO—X—(CH₂)_(m1)—X—CO—,    -   —X—(CH₂)_(m1)—X—CO—(CH₂)_(m2)—,    -   —X—(CH₂)_(m1)—CO—X—(CH₂)_(m2)—,    -   —X—(CH₂)_(m1)—X—CO—(CH₂)_(m2)—X—,    -   —X—(CH₂)_(m1)—X—CO—(CH₂)_(m2)—CO—,    -   —X—(CH₂)_(m1)—CO—X—(CH₂)_(m2)—X—, and    -   —X—(CH₂)_(m1)—CO—X—(CH₂)_(m2)—CO—;

wherein;

-   -   each X is independently selected from —O—, —S—, or —NH— or is        missing;    -   each m₁ is independently 0 to 5;    -   each m₂ is independently 1 to 5;    -   and wherein the linker is attached to the N-terminal amino group        of C-peptide.

In another aspect of any of these branched chain PEGylated C-peptides,the PEGylated C-peptide comprises a linker connecting the PEG moiety toC-peptide selected from;

-   -   —X₁—(CH₂)_(m4)—CO—    -   —X₁—CO—;    -   —X₁—CO—(CH₂)_(m4)—CO—    -   —X₁—CO—X₂—(CH₂)_(m3)—CO—; and    -   X₁—(CH₂)_(m2)—X₂—CO—(CH₂)_(m4)—CO—;

wherein;

-   -   X₁ is —O—, or missing;    -   X₂ is —NH—;    -   m₂ is 1 to 5;    -   m₃ is 2; and    -   m₄ is 1 to 5.

In another aspect of any of these branched chain PEGylated C-peptides,the PEGylated C-peptide comprises a linker connecting the PEG moiety toC-peptide selected from;

-   -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—X—,    -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—(CH₂)_(m5)—CO—,    -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—CO—, and    -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—CO—(CH₂)_(m5)—CO—;

wherein;

-   -   X is independently selected from —O—, —S—, or —NH— or is        missing;    -   X₁ is —O—, or missing;    -   X₂ is —NH—;    -   each m₅ is independently selected from 1 to 5; and    -   each n₃ is independently selected from 1 to 400.

In another aspect of any of these branched chain PEGylated C-peptides,the PEGylated C-peptide comprises a linker connecting the PEG moiety toC-peptide selected from;

-   -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—(CH₂)_(m6)—CO—,    -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—CO—, and    -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—CO—(CH₂)_(m7)—CO—;

wherein;

-   -   X₁ is —O—, or is missing;    -   X₂ is —NH—;    -   m₅ is 3;    -   m₆ is independently 2 or 5;    -   m₇ is 3; and    -   n₃ is 1 to 400.

In another embodiment, the present invention includes a PEGylatedC-peptide wherein the PEGylated C-peptide has the structure:

wherein;

-   -   R₁=alkyl;    -   n₁ is 200 to 800;    -   n₂ is 200 to 800.

In another embodiment, the present invention includes a PEGylatedC-peptide wherein the PEGylated C-peptide has the structure:

wherein;

-   -   R₁=alkyl;    -   n₁ is 200 to 800; and    -   n₂ is 200 to 800.

In another embodiment, the present invention includes a PEGylatedC-peptide wherein the PEGylated C-peptide has the structure:

wherein;

-   -   R₁=alkyl;    -   n₁ is 200 to 800;    -   n₂ is 200 to 800; and    -   n₃ is 1 to 400.

In another embodiment, the present invention includes a PEGylatedC-peptide wherein the PEGylated C-peptide has the structure:

wherein;

-   -   R₁=alkyl;    -   n₁ is 200 to 800;    -   n₂ is 200 to 800; and    -   n₃ is 1 to 400.

In another embodiment, the present invention includes a PEGylatedC-peptide wherein the PEGylated C-peptide has the structure:

wherein;

-   -   R₁=alkyl;    -   n₁ is 200 to 800;    -   n₂ is 200 to 800; and    -   n₃ is 1 to 400.

In another aspect, the PEGylated C-peptide comprises a branched chainPEG of general formula (III):

wherein;

-   -   R₁=alkyl;    -   n₁ is 20 to 800;    -   n₂ is 20 to 800; and    -   wherein each linker is independently defined as below, and    -   wherein the linker connecting to C-peptide is attached to the        N-terminal amino group of C-peptide.

In one embodiment of the PEGylated C-peptides of formula (III), thePEGylated C-peptide has the structure (III A):

-   -   wherein;        -   R₁=alkyl;        -   n₁ is 20 to 800;        -   n₂ is 20 to 800; and        -   wherein the linker is defined as below, and is attached to            the N-terminal amino group of C-peptide.

In another embodiment, the present invention includes a PEGylatedC-peptide wherein the PEGylated C-peptide has the structure:

-   -   wherein;        -   R₁=alkyl;        -   n₁ is 200 to 800; and        -   n₂ is 200 to 800;

In another aspect, the PEGylated C-peptide comprises a branched chainPEG of general formula (IV):

wherein;

-   -   R₁=alkyl;    -   n₁ is 20 to 800;    -   n₂ is 20 to 800;    -   wherein each linker is independently defined as below;    -   wherein the linker connecting the lysine residue to C-peptide is        attached to the N-terminal amino group of C-peptide and the        C-terminal carboxylate group of the lysine residue, and wherein        the linkers connecting the lysine moiety to the PEG moieties are        linked through the amino groups of the lysine molecule.

In another embodiment, the present invention includes a PEGylatedC-peptide wherein the PEGylated C-peptide has the structure:

wherein;

-   -   R₁=alkyl;    -   n₁ is 200 to 800; and    -   n₂ is 200 to 800.

In certain aspects of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 5-fold greater than unmodified C-peptide when subcutaneouslyadministered to dogs.

In certain aspects of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 6-fold greater than unmodified C-peptide when subcutaneouslyadministered to dogs.

In certain aspects of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 7-fold greater than unmodified C-peptide when subcutaneouslyadministered to dogs.

In certain aspects of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 8-fold greater than unmodified C-peptide when subcutaneouslyadministered to dogs.

In certain aspects of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 10-fold greater than unmodified C-peptide whensubcutaneously administered to dogs.

In certain aspects of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 15-fold greater than unmodified C-peptide whensubcutaneously administered to dogs.

In certain aspects of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 20-fold greater than unmodified C-peptide whensubcutaneously administered to dogs.

In certain aspects of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 25-fold greater than unmodified C-peptide whensubcutaneously administered to dogs.

In certain aspects of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 50-fold greater than unmodified C-peptide whensubcutaneously administered to dogs.

In certain aspects of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 75-fold greater than unmodified C-peptide whensubcutaneously administered to dogs.

In certain aspects of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 100-fold greater than unmodified C-peptide whensubcutaneously administered to dogs.

In certain aspects of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has an equi-potent biological activity with theunmodified C-peptide. In certain aspects of any of the claimed PEGylatedC-peptides, the PEGylated C-peptide retains at least about 95% of thebiological activity of the unmodified C-peptide. In certain aspects ofany of the claimed PEGylated C-peptides, the PEGylated C-peptide retainsat least about 90% of the biological activity of the unmodifiedC-peptide. In certain aspects of any of the claimed PEGylatedC-peptides, the PEGylated C-peptide retains at least about 80% of thebiological activity of the unmodified C-peptide. In another aspect ofany of the claimed PEGylated C-peptides, the PEGylated C-peptide retainsat least about 70% of the biological activity of the unmodifiedC-peptide. In another aspect of any of the claimed PEGylated C-peptides,the PEGylated C-peptide retains at least about 60% of the biologicalactivity of the unmodified C-peptide. In another aspect of any of theclaimed PEGylated C-peptides, the PEGylated C-peptide retains at leastabout 50% of the biological activity of the unmodified C-peptide. Inanother aspect of any of the claimed PEGylated C-peptides, the PEGylatedC-peptide retains at least about 40% of the biological activity of theunmodified C-peptide. In another aspect of any of the claimed PEGylatedC-peptides, the PEGylated C-peptide retains at least about 30% of thebiological activity of the unmodified C-peptide. In another aspect ofany of the claimed PEGylated C-peptides, the PEGylated C-peptide retainsat least about 20% of the biological activity of the unmodifiedC-peptide. In another aspect of any of the claimed PEGylated C-peptides,the PEGylated C-peptide retains at least about 10% of the biologicalactivity of the unmodified C-peptide. In another aspect of any of theclaimed PEGylated C-peptides, the PEGylated C-peptide retains at leastabout 5% of the biological activity of the unmodified C-peptide.

In another embodiment, the present invention includes a dosing regimenwhich maintains an average steady-state concentration of PEGylatedC-peptide in the patient's plasma of between about 0.2 nM and about 6 nMwhen using a dosing interval of 3 days or longer, comprisingadministering to the patient a therapeutic dose of PEGylated C-peptideof any of the claimed PEGylated C-peptides.

In another embodiment, the present invention includes a dosing regimenwhich maintains an average steady-state concentration of PEGylatedC-peptide in the patient's plasma of between about 0.4 nM and about 6 nMwhen using a dosing interval of 3 days or longer, comprisingadministering to the patient a therapeutic dose of PEGylated C-peptideof any of the claimed PEGylated C-peptides.

In another embodiment, the present invention includes a dosing regimenwhich maintains an average steady-state concentration of PEGylatedC-peptide in the patient's plasma of between about 0.6 nM and about 8 nMwhen using a dosing interval of 3 days or longer, comprisingadministering to the patient a therapeutic dose of PEGylated C-peptideof any of the claimed PEGylated C-peptides.

In another embodiment, the present invention includes a dosing regimenwhich maintains an average steady-state concentration of PEGylatedC-peptide in the patient's plasma of between about 0.8 nM and about 10nM when using a dosing interval of 3 days or longer, comprisingadministering to the patient a therapeutic dose of PEGylated C-peptideof any of the claimed PEGylated C-peptides.

In another embodiment, the present invention includes a method formaintaining C-peptide levels above the minimum effective therapeuticlevel in a patient in need thereof, comprising administering to thepatient a therapeutic dose of any of the claimed PEGylated C-peptides.

In another aspect of any of the claimed PEGylated C-peptides, thePEGylated C-peptide is substantially free of adverse side effects whensubcutaneously administered to a mammal at an effective therapeuticdose.

In another embodiment, the present invention includes a method fortreating one or more long-term complications of diabetes in a patient inneed thereof, comprising administering to the patient a therapeutic doseof any of the claimed PEGylated C-peptides.

In another embodiment, the present invention includes a method fortreating a patient with diabetes comprising administering to the patienta therapeutic dose of PEGylated C-peptide of any of the claimedPEGylated C-peptides in combination with insulin.

In one aspect of any of these methods, the PEGylated C-peptide isadministered with a dosing interval of about 3 days or longer. In oneaspect of any of these methods, the PEGylated C-peptide is administeredwith a dosing interval of about 4 days or longer. In one aspect of anyof these methods, the PEGylated C-peptide is administered with a dosinginterval of about 5 days or longer. In one aspect of any of thesemethods, the PEGylated C-peptide is administered with a dosing intervalof about 6 days or longer. In one aspect of any of these methods, thePEGylated C-peptide is administered with a dosing interval of about 7days or longer.

In certain embodiments, treatment results in an improvement of at least10 in nerve conduction velocity compared to nerve conduction velocityprior to starting PEGylated C-peptide therapy.

In another aspect of any of these methods, the plasma concentration ofPEGylated C-peptide is maintained above about 0.1 nM. In another aspectof any of these methods, the plasma concentration of PEGylated C-peptideis maintained above about 0.2 nM. In another aspect of any of thesemethods, the plasma concentration of PEGylated C-peptide is maintainedabove about 0.3 nM. In another aspect of any of these methods, theplasma concentration of PEGylated C-peptide is maintained above about0.4 nM.

In another aspect of any of these methods, the therapeutic dose ofPEGylated C-peptide is administered subcutaneously. In another aspect ofany of these methods, the therapeutic dose of PEGylated C-peptide isadministered orally.

In another embodiment, the present invention includes the use of any ofthe claimed PEGylated C-peptides as a C-peptide replacement therapy in apatient in need thereof.

In another embodiment, the present invention includes the use of any ofthe claimed PEGylated C-peptides for treating one or more long-termcomplications of diabetes in a patient in need thereof. In certainembodiments, the long-term complications of diabetes are selected fromthe group consisting of retinopathy, peripheral neuropathy, autonomicneuropathy, and nephropathy. In certain embodiments, the long-termcomplication of diabetes is peripheral neuropathy. In certainembodiments, the peripheral neuropathy is established peripheralneuropathy. In certain embodiments, treatment results in an improvementof at least 10% in nerve conduction velocity compared to nerveconduction velocity prior to starting PEGylated C-peptide therapy.

In another embodiment, the present invention includes a pharmaceuticalcomposition comprising any of the claimed PEGylated C-peptides and apharmaceutically acceptable carrier or excipient. In certainembodiments, the pharmaceutically acceptable carrier or excipient issorbitol. In certain embodiments, the sorbitol is present at aconcentration of about 2% to about 8% wt/wt. In certain embodiments, thesorbitol is present at a concentration of about 4.7%. In certainembodiments, the pharmaceutical composition is buffered to a pH withinthe range of about pH 5.5 to about pH 6.5. In certain embodiments, thepharmaceutical composition is buffered to a pH of about 6.0. In certainembodiments, the pharmaceutical composition is buffered with a phosphatebuffer at a concentration of about 5 mM to about 25 mM. In certainembodiments, the pharmaceutical composition is buffered with a phosphatebuffer at a concentration of about 10 mM. In one aspect of any of theseembodiments, the pharmaceutical composition is characterized by improvedstability of any of the claimed PEGylated C-peptides compared to apharmaceutical composition comprising the same PEGylated C-peptide and0.9% saline at pH 7.0, wherein the stability is determined afterincubation for a predetermined time at 40° C. In different embodiments,the pre-determined time is about one week, about 2 weeks, about threeweeks, about four weeks, or about five weeks, or about six weeks.

In another embodiment, the present invention includes a pharmaceuticalcomposition comprising any of the claimed PEGylated C-peptides andinsulin.

Certain embodiments include the use of any of the disclosed PEGylatedC-peptides to reduce the risk of hypoglycemia in a human patient withinsulin dependent diabetes, in a regimen which additionally comprisesthe administration of insulin, comprising; a) administering insulin tothe patient; b) administering a therapeutic dose of the PEGylatedC-peptide in a different site as that used for the patient's insulinadministration; c) adjusting the dosage amount, type, or frequency ofinsulin administered based on the patient's altered insulin requirementsresulting from the therapeutic dose of the PEGylated C-peptide.

In some embodiments, the patient has at least one long termcomplications of diabetes.

Certain embodiments include a method for treating an insulin-dependenthuman patient, comprising the steps of; a) administering insulin to thepatient, wherein the patient has neuropathy; b) administeringsubcutaneously to the patient a therapeutic dose of any of the disclosedPEGylated C-peptides in a different site as that used for the patient'sinsulin administration; c) adjusting the dosage amount, type, orfrequency of insulin administered based on monitoring the patient'saltered insulin requirements resulting from the therapeutic dose ofPEGylated C-peptide, wherein the adjusted dose of insulin reduces therisk, incidence, or severity of hypoglycemia, wherein the adjusted doseof insulin is at least 10% less than the patient's insulin dose prior tostarting PEGylated C-peptide treatment.

Certain embodiments include a method of reducing insulin usage in aninsulin-dependent human patient, comprising the steps of; a)administering insulin to the patient; b) administering subcutaneously tothe patient a therapeutic dose any of the disclosed PEGylated C-peptidesin a different site as that used for the patient's insulinadministration; c) adjusting the dosage amount, type, or frequency ofinsulin administered based on monitoring the patient's altered insulinrequirements resulting from the therapeutic dose of PEGylated C-peptide,wherein the adjusted dose of insulin does not induce hypoglycemia,wherein the adjusted dose of insulin is at least 10% less than thepatient's insulin dose prior to starting the PEGylated C-peptidetreatment.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the presentinvention can be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the invention are utilized, and the accompanying drawingsof which:

FIG. 1 shows a reverse phase chromatogram of a 40 kDa branched chainPEGylated C-peptide of the invention.

FIG. 2 shows a size exclusion chromatogram of a 40 kDa branched chainPEGylated C-peptide of the invention.

FIG. 3 shows a reverse phase chromatogram of a 20 kDa linear chainPEGylated C-peptide of the invention.

FIG. 4 shows a size exclusion chromatogram of a 20 kDa linear chainPEGylated C-peptide of the invention.

FIG. 5 shows the plasma concentration-time profiles of unmodifiedC-peptide in dogs following a single subcutaneous dose. FIG. 5A showsthe profile on a time scale of one day, FIG. 5B shows the profile on atime scale of 12 days.

FIG. 6 shows the plasma concentration-time profiles of C-peptide in dogsfollowing single subcutaneous doses of the 20 kDa linear chain PEGylatedC-peptide (diamonds) and the 40 kDa branched chain PEGylated C-peptide(squares) using a linear scale.

FIG. 7 shows the plasma concentration-time profiles of C-peptide in dogsfollowing single subcutaneous doses of the 20 kDa linear chain PEGylatedC-peptide (diamonds) and the 40 kDa branched chain PEGylated C-peptide(squares) using a semi-logarithmic scale.

FIG. 8 shows the plasma concentration-time profiles of C-peptide in dogsfollowing single subcutaneous doses of PEGylated C-peptide. FIG. 8Ashows data presented using a linear scale. FIG. 8B shows data presentedin semi-logarithmic form.

FIG. 9 shows C_(max) and AUC_((0-t)) of C-peptide in dogs followingsingle subcutaneous doses of PEGylated C-peptide. FIG. 9A shows C_(max)and FIG. 9B shows AUC_((0-t)).

FIG. 10 shows Mean (±SD) C-peptide plasma concentration-time profile inSprague Dawley rats following single-dose subcutaneous administration ofPEGylated C-peptide. Panel A Linear scale; Panel B Semi-log scale.

FIG. 11 shows mean (±SD) C-peptide plasma concentration-time profile inCynomolgus monkeys following single-dose subcutaneous administration ofPEGylated C-peptide. Panel A Linear scale; Panel B Semi-log scale.

FIG. 12 shows mean C-peptide plasma concentration-time profile inSprague Dawley rats following multiple dose subcutaneous administrationof PEGylated C-peptide. Panel A Male, Linear scale; Panel B FemaleLinear scale; Panel C Male Semi-log scale; Panel D Female Semi-logscale.

FIG. 13 shows the relationship between C_(max) and AUC_((0-inf)) inSprague Dawley rats as a function of dose; Panel A C_(max), first dose;Panel B AUC_((0-inf)) first dose; Panel C C_(max), last dose; Panel DAUC_((0-inf)) last dose.

FIG. 14 shows Mean C-peptide plasma concentration-time profile inCynomolgus monkeys upon multiple-dose subcutaneous administration PanelA Male, Linear scale; Panel B Female Linear scale; Panel C Male Semi-logscale; Panel D Female Semi-log scale.

FIG. 15 shows the relationship between C_(max) and AUC_((0-inf)) inCynomolgus monkeys as a function of dose; Panel A C_(max), first dose;Panel B AUC_((0-inf)) first dose; Panel C C_(max), last dose; Panel DAUC_((0-inf)) last dose.

FIG. 16 shows the caudal Nerve Conduction Velocity (NCV) in each of fourtreatment groups of rats treated for up to 8 weeks (See Example 6). Inthis figure, and the following three figures, Group 1 represents thevehicle control (no streptozotocin [STZ]), Group 2 represents the STZtreated group plus vehicle, Group 3 represents STZ plus 1.0 mg/kg/week(1.0 mg/ml) of human PEGylated C-peptide (Example 12), and Group 4represents STZ plus 3.0 mg/kg/week (1.0 mg/ml) of human PEGylatedC-peptide (Example 12). In FIG. 16, panel A shows the baseline NCVmeasurements, and panel B shows caudal NCV after a 4-week period (fromBaseline) of administration of either vehicle alone or the PEGylatedC-peptide at either 1.0 or 3.0 mg/kg/week. FIG. 16, panel C shows caudalNCV after an 8-week period (from Baseline) of administration of eithervehicle alone or the PEGylated C-peptide at either 1.0 or 3.0mg/kg/week.

FIG. 17 shows the digital NCV in each of the same four groups of animalsas described in FIG. 16. In FIG. 17, panel A shows the baselinemeasurements, and panel B shows digital NCV after a 4-week period (fromBaseline) of administration of either vehicle alone or the PEGylatedC-peptide at either 1.0 or 3.0 mg/kg/week. FIG. 17, panel C showsdigital NCV after an 8-week period (from Baseline) of administration ofeither vehicle alone or the PEGylated C-peptide at either 1.0 or 3.0mg/kg/week.

FIG. 18 shows the relative change in digital NCV in the same 4 treatmentgroups as described in FIG. 17, over the entire duration of the study.

FIG. 19 shows the relative change in caudal and digital NCV in the same4 treatment groups as described in FIGS. 16 and 17, compared to baselinemeasurements after 8 weeks of treatment.

FIG. 20 shows a Fourier Transform Infrared Spectroscopy (FT-IR): ofC-peptide, the PEG reagent, and PEGylated C-peptide.

FIG. 21 shows an expanded region of the Fourier Transform InfraredSpectroscopy (FT-IR): of C-peptide, the PEG reagent, and PEGylatedC-peptide.

FIG. 22 shows a Fourier Transform Infrared Spectroscopy (FT-IR): ofC-peptide, the PEG reagent, and PEGylated C-peptide collected in D₂O.

FIG. 23 shows a Fourier Transform Infrared Spectroscopy (FT-IR): ofC-peptide, the PEG reagent, and PEGylated C-peptide collected in D₂O.

FIG. 24 shows a peptide map for C-peptide (1 mg/mL) and PEGylatedC-peptide (10 mg/mL) after incubation with chymotrypsin.

FIG. 25 shows the normalized sedimentation coefficient distribution forPEGylated C-peptide (at ˜0.6 mg/mL) in PBS buffer.

FIG. 26 shows a Circular Dichroism Analysis of C-peptide and PEGylatedC-peptide.

FIG. 27 shows the results of Size Exclusion Chromatography (SEC) of asample of the PEGylated C-peptide of Example 12.

FIG. 28 shows an overlay of the chromatogram of the 20 kDa PEGylatedC-peptide and 40 kDa PEGylated C-peptide of Example 12.

FIG. 29 shows the results of sodium dodecyl sulfate polyacrylamide gelelectrophoresis SDS-PAGE: Gel electrophoresis of the PEGylated C-peptideof Example 12.

FIG. 30 shows the results of an assessment of the biological activity ofthe PEGylated C-peptide compared to native C-peptide in the ERKphosphorylation assay.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “active” or “activated” when used in conjunction with aparticular functional group refers to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react(i.e., a “non-reactive” or “inert” group). As used herein, the term“functional group” or any synonym thereof is meant to encompassprotected forms thereof as well as unprotected forms.

The term “alkoxy” refers to an —O—R group, wherein R is alkyl orsubstituted alkyl, preferably C1-6 alkoxy (e.g., methoxy, ethoxy,propyloxy, and so forth).

The term “alkyl” refers to a hydrocarbon, typically ranging from about 1to 12 atoms in length. Hydrocarbons may be branched or linear and arepreferably, but not necessarily saturated. Exemplary alkyl groupsinclude methyl, ethyl, propyl, butyl, pentyl, 2-methylbutyl,2-ethylpropyl, etc. As used herein “alkyl” includes cycloalkyl as wellas cycloalkylene alkyls. The term “lower alkyl” refers to an alkyl groupcontaining from 1 to 6 carbon atoms, and may be straight chain orbranched.

The term “C_(max)” as used herein is the maximum serum or plasmaconcentration of drug which occurs during the period of release which ismonitored.

The term “C_(min)” as used herein is the minimum serum or plasmaconcentration of drug which occurs during the period of release duringthe treatment period.

The term “C_(ave)” as used herein is the average serum or plasmaconcentration of drug derived by dividing the area under the curve (AUC)of the release profile by the duration of the release.

The term “C_(ss-ave)” as used herein is the average steady-stateconcentration of drug obtained during a multiple dosing regimen afterdosing for at least five elimination half-lives. It will be appreciatedthat drug concentrations are fluctuating within dosing intervals evenonce an average steady-state concentration of drug has been obtained.

The term “t_(max)” as used herein is the time post-dose at which C_(max)is observed.

The term “AUC” as used herein means “area under curve” for the serum orplasma concentration-time curve, as calculated by the trapezoidal ruleover the complete sample collection interval.

The term “bioavailability” refers to the amount of drug that reaches thecirculation system expressed in percent of that administered. The amountof bioavailable material can be defined as the calculated AUC for therelease profile of the drug during the time period starting atpost-administration and ending at a predetermined time point. As isunderstood in the art, a release profile is generated by graphing theserum levels of a biologically active agent in a subject (Y-axis) atpredetermined time points (X-axis). Bioavailability is often referred toin terms of % bioavailability, which is the bioavailability achieved fora drug (such as C-peptide) following administration of a sustainedrelease composition of that drug divided by the bioavailability achievedfor the drug following intravenous administration of the same equivalentdose of the drug, multiplied by 100.

The phrase “conservative amino acid substitution” or “conservativemutation” refers to the replacement of one amino acid by another aminoacid with a common property. A functional way to define commonproperties between individual amino acids is to analyze the normalizedfrequencies of amino acid changes between corresponding proteins ofhomologous organisms (Schulz G E and RH Schirmer, Principles of ProteinStructure, Springer-Verlag (1979)). According to such analyses, groupsof amino acids can be defined where amino acids within a group exchangepreferentially with each other, and therefore resemble each other mostin their impact on the overall protein structure (Schulz G E and RHSchirmer, Principles of Protein Structure, Springer-Verlag (1979)).

Examples of amino acid groups defined in this manner include: a“charged/polar group,” consisting of Glu, Asp, Asn, Gln, Lys, Arg, andHis; an “aromatic or cyclic group,” consisting of Pro, Phe, Tyr, andTrp; and an “aliphatic group,” consisting of Gly, Ala, Val, Leu, Ile,Met, Ser, Thr, and Cys.

Within each group, subgroups can also be identified, e.g., the group ofcharged/polar amino acids can be sub-divided into the subgroupsconsisting of the “positively-charged subgroup,” consisting of Lys, Arg,and His; the “negatively-charged subgroup,” consisting of Glu and Asp,and the “polar subgroup” consisting of Asn and Gln. The aromatic orcyclic group can be sub-divided into the subgroups consisting of the“nitrogen ring subgroup,” consisting of Pro, His, and Trp; and the“phenyl subgroup” consisting of Phe and Tyr. The aliphatic group can besub-divided into the subgroups consisting of the “large aliphaticnon-polar subgroup,” consisting of Val, Leu, and Ile; the “aliphaticslightly-polar subgroup,” consisting of Met, Ser, Thr, and Cys; and the“small-residue sub-group,” consisting of Gly and Ala.

Examples of conservative mutations include amino acid substitutions ofamino acids within the subgroups above, e.g., Lys for Arg and vice versasuch that a positive charge can be maintained; Glu for Asp and viceversa such that a negative charge can be maintained; Ser for Thr suchthat a free —OH can be maintained; and Gln for Asn such that a free —NH₂can be maintained. “Semi-conservative mutations” include amino acidsubstitutions of amino acids with the same groups listed above, which donot share the same subgroup. For example, the mutation of Asp for Asn,or Asn for Lys, all involve amino acids within the same group, butdifferent subgroups. “Non-conservative mutations” involve amino acidsubstitutions between different groups, e.g., Lys for Leu, Phe for Ser.

The terms “Dalton”, “Da”, or “D” refers to an arbitrary unit of mass,being 1/12 the mass of the nuclide of carbon-12, equivalent to1.657×10⁻²⁴ g. The term “kDa” is for kilodalton (i.e., 1000 Daltons).

The terms “diabetes”, “diabetes mellitus”, or “diabetic condition”,unless specifically designated otherwise, encompass all forms ofdiabetes. The term “type 1 diabetic” or “type 1 diabetes” refers to apatient with a fasting plasma glucose concentration of greater thanabout 7.0 mmoL/L and a fasting C-peptide level of about, or less thanabout 0.2 nmoL/L. The term “type 1.5 diabetic” or “type 1.5 diabetes”refers to a patient with a fasting plasma glucose concentration ofgreater than about 7.0 mmoL/L and a fasting C-peptide level of about, orless than about 0.4 nmoL/L. The term “type 2 diabetic” or “type 2diabetes” generally refers to a patient with a fasting plasma glucoseconcentration of greater than about 7.0 mmoL/L and fasting C-peptidelevel that is within or higher than the normal physiological range ofC-peptide levels (about 0.47 to 2.5 nmoL/L). It will be appreciated thata patient initially diagnosed as a type 2 diabetic may subsequentlydevelop insulin-dependent diabetes, and may remain diagnosed as a type 2patient, even though their C-peptide levels drop to those of a type 1.5or type 1 diabetic patient (<0.2 nmol/L).

The terms “insulin-dependent patient” or “insulin-dependent diabetes”encompass all forms of diabetics/diabetes who/that require insulinadministration to adequately maintain normal glucose levels unlessspecified otherwise.

Diabetes is frequently diagnosed by measuring fasting blood glucose,insulin, or glycated hemoglobin levels (which are typically referred toas hemoglobin A1c, Hb_(1c), Hb_(A1c), or A1C). Normal adult glucoselevels are 60-126 mg/dL. Normal insulin levels are 30-60 pmoL/L. NormalHbA1c levels are generally less than 6%. The World Health Organizationdefines the diagnostic value of fasting plasma glucose concentration to7.0 mmoL/L (126 mg/dL) and above for diabetes mellitus (whole blood 6.1mmoL/L or 110 mg/dL), or 2-hour glucose level greater than or equal to11.1 mmoL/L (greater than or equal to 200 mg/dL). Other valuessuggestive of or indicating high risk for diabetes mellitus includeelevated arterial pressure greater than or equal to 140/90 mm Hg;elevated plasma triglycerides (greater than or equal to 1.7 mmoL/L [150mg/dL]) and/or low HDL-cholesterol (less than 0.9 mmoL/L [35 mg/dL] formen; and less than 1.0 mmoL/L [39 mg/dL] for women); central obesity(BMI exceeding 30 kg/m²); microalbuminuria, where the urinary albuminexcretion rate is greater than or equal to 20 μg/min or the albumincreatinine ratio is greater than or equal to 30 mg/g.

The term “delivery agent” refers to carrier compounds or carriermolecules that are effective in the oral delivery of therapeutic agents,and may be used interchangeably with “carrier”.

The term “homology” describes a mathematically-based comparison ofsequence similarities which is used to identify genes or proteins withsimilar functions or motifs. The nucleic acid and protein sequences ofthe present invention can be used as a “query sequence” to perform asearch against public databases to, e.g., identify other family members,related sequences, or homologs. Such searches can be performed using theNBLAST and XBLAST programs (version 2.0) of Altschul et al.: J. Mol.Biol. 215: 403-410, (1990). BLAST nucleotide searches can be performedwith the NBLAST program, score=100, wordlength=12 to obtain nucleotidesequences homologous to nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.:Nucleic Acids Res. 25(17): 3389-3402, (1997). When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and BLAST) can be used (see www.ncbi.nlm.nih.gov).

The term “homologous” refers to the relationship between two proteinsthat possess a “common evolutionary origin”, including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) in the same speciesof animal, as well as homologous proteins from different species ofanimal (e.g., myosin light chain polypeptide; see Reeck et al.: Cell 50:667, (1987)). Such proteins (and their encoding nucleic acids) havesequence homology, as reflected by their sequence similarity, whether interms of percent identity or by the presence of specific residues ormotifs and conserved positions. In specific embodiments, two nucleicacid sequences are “substantially homologous” or “substantially similar”when at least about 85%, and more preferably at least about 90 or atleast about 95% of the nucleotides match over a defined length of thenucleic acid sequences, as determined by a sequence comparison algorithmknown such as BLAST, FASTA, DNA Strider, CLUSTAL, etc. An example ofsuch a sequence is an allelic or species variant of the specific genesof the present invention. Sequences that are substantially homologousmay also be identified by hybridization, e.g., in a Southernhybridization experiment under, e.g., stringent conditions as definedfor that particular system.

Similarly, in particular embodiments of the invention, two amino acidsequences are “substantially homologous” or “substantially similar” whengreater than 80 of the amino acid residues are identical, or whengreater than about 90% of the amino acid residues are similar (i.e., arefunctionally identical). Preferably the similar or homologouspolypeptide sequences are identified by alignment using, e.g., the GCG(Genetics Computer Group, version 7, Madison, Wis.) pileup program, orusing any of the programs and algorithms described above. The programmay use the local homology algorithm of Smith and Waterman with thedefault values: gap creation penalty=−(1+⅓ k), k being the gap extensionnumber, average match=1, average mismatch=−0.333.

As used herein, “identity” means the percentage of identical nucleotideor amino acid residues at corresponding positions in two or moresequences when the sequences are aligned to maximize sequence matching,i.e., taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in Computational Molecular Biology, Lesk A M, Ed., OxfordUniversity Press, New York, (1988); Biocomputing: Informatics and GenomeProjects, Smith D W, Ed., Academic Press, New York, (1993); ComputerAnalysis of Sequence Data, Part I, Griffin A M and Griffin H G, Eds.,Humana Press, New Jersey, (1994); Sequence Analysis in MolecularBiology, von Heinje G, Academic Press, (1987); and Sequence AnalysisPrimer, Gribskov M and Devereux J, Eds., M Stockton Press, New York,(1991); and Carillo H and Lipman D, SIAM J. Applied Math., 48: 1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux J et al.: NucleicAcids Res. 12(1): 387, (1984)), BLASTP, BLASTN, and FASTA (Altschul S Fet al.: J. Molec. Biol. 215: 403-410, (1990) and Altschul S F et al.:Nucleic Acids Res. 25: 3389-3402, (1997)). The BLAST X program ispublicly available from NCBI and other sources (BLAST Manual, Altschul SF et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul S F et al., J. Mol.Biol. 215: 403-410, (1990)). The well-known Smith Waterman algorithm(Smith T F, Waterman M S: J. Mol. Biol. 147(1): 195-197, (1981)) canalso be used to determine similarity between sequences.

The term “insulin” includes all forms of insulin including, withoutlimitation, rapid-acting forms, such as Insulin Lispro rDNA origin:HUMALOG (1.5 mL, 10 mL, Eli Lilly and Company, Indianapolis, Ind.),Insulin Injection (Regular Insulin) from beef and pork (regular ILETINI, Eli Lilly), human: rDNA: HUMULIN R (Eli Lilly), NOVOLIN R (NovoNordisk, New York, N.Y.), Semi synthetic: VELOSULIN Human (NovoNordisk), rDNA Human, Buffered: VELOSULIN BR, pork: regular Insulin(Novo Nordisk), purified pork: Pork Regular ILETIN II (Eli Lilly),Regular Purified Pork Insulin (Novo Nordisk), and Regular (Concentrated)ILETIN II U-500 (500 units/mL, Eli Lilly); intermediate-acting formssuch as Insulin Zinc Suspension, beef and pork: LENTE ILETIN G I (EliLilly), Human, rDNA: HUMULIN L (Eli Lilly), NOVOLIN L (Novo Nordisk),purified pork: LENTE ILETIN II (Eli Lilly), Isophane Insulin Suspension(NPH): beef and pork: NPH ILETIN I (Eli Lilly), Human, rDNA: HUMULIN N(Eli Lilly), Novolin N (Novo Nordisk), purified pork: Pork NPH Eetin II(Eli Lilly), NPH-N (Novo Nordisk); and long-acting forms such as Insulinzinc suspension, extended (ULTRALENTE, Eli Lilly), human, rDNA: HUMULINU (Eli Lilly).

The terms “measuring” or “measurement” mean assessing the presence,absence, quantity, or amount (which can be an effective amount) ofeither a given substance within a clinical- or patient-derived sample,including the derivation of qualitative or quantitative concentrationlevels of such substances, or otherwise evaluating the values orcategorization of a patient's clinical parameters.

The term “meal” as used herein means a standard and/or a mixed meal.

The term “mean”, when preceding a pharmacokinetic value (e.g., meant_(max)), represents the arithmetic mean value of the pharmacokineticvalue unless otherwise specified.

The term “mean baseline level” as used herein means the measurement,calculation, or level of a certain value that is used as a basis forcomparison, which is the mean value over a statistically significantnumber of subjects, e.g., across a single clinical study or acombination of more than one clinical study.

The term “multiple dose” means that the patient has received at leasttwo doses of the drug composition in accordance with the dosing intervalfor that composition.

The term “neuropathy” in the context of a “patient with neuropathy” or apatient that “has neuropathy”, means that the patient meets at least oneof the four criteria outlined in the San Antonio Conference on diabeticneuropathy (report and recommendations of the San Antonio Conference ondiabetic neuropathy. Ann. Neurol. 24 99-104 (1988)), which in briefinclude 1) clinical signs of polyneuropathy, 2) symptoms of nervedysfunction, 3) nerve conduction deficits in at least two nerves, or 4)quantitative sensory deficits. The term “established neuropathy” meansthat the patient meets at least two of the four criteria outlined in theSan Antonio Conference on diabetic neuropathy. The term “incipientneuropathy” refers to a patient that exhibits only nerve conductiondeficits, and no other symptoms of neuropathy.

The term “normal glucose levels” is used interchangeably with the term“normoglycemic” and “normal” and refers to a fasting venous plasmaglucose concentration of less than about 6.1 mmoL/L (110 mg/dL).Sustained glucose levels above normoglycemic are considered apre-diabetic condition.

As used herein, the term “patient” in the context of the presentinvention is preferably a mammal. The mammal can be a human, non-humanprimate, mouse, rat, dog, cat, horse, or cow, but are not limited tothese examples. Mammals other than humans can be advantageously used aspatients that represent animal models of insulin-dependent diabetesmellitus, or diabetic conditions. A patient can be male or female. Apatient can be one who has been previously diagnosed or identified ashaving insulin-dependent diabetes, or a diabetic condition, andoptionally has already undergone, or is undergoing, a therapeuticintervention for the diabetes. A patient can also be one who issuffering from a long-term complication of diabetes. Preferably thepatient is human.

The terms “PEG”, “polyethylene glycol”, or “poly(ethylene glycol)” asused herein refers to any water soluble poly(ethylene oxide), andincludes molecules comprising the structure —(CH₂CH₂O)_(n)— where n isan integer from 2 to about 800. A commonly used PEG is end-capped PEG,wherein one end of the PEG is capped with a relatively inactive groupsuch as an alkoxy while the other end is a hydroxyl group that may befurther modified. An often-used capping group is methoxy and thecorresponding end-capped PEG is often denoted mPEG. The notion PEG isoften used instead of mPEG. Specific PEG forms of the invention arebranched, linear, forked PEGs, and the like and the PEG groups aretypically polydisperse, possessing a low polydispersity index of lessthan about 1.05. The PEG moieties of the invention will for a givenmolecular weight will typically consist of a range of ethylene glycol(or ethyleneoxide) monomers. For example, A PEG moiety of molecularweight 2000 Da will typically consist of 43±10 monomers, the averagebeing around 43 monomers. The term “PEGylated” refers to the covalentattachment of PEG to another molecule, such as C-peptide.

The term “replacement dose” in the context of a replacement therapy forC-peptide refers to a dose of C-peptide or PEGylated C-peptide thatmaintains C-peptide or PEGylated C-peptide levels in the blood within adesirable range, particularly at a level which is at or above theminimum effective therapeutic level. In certain aspects, the replacementdose maintains the average steady-state concentration C-peptide orPEGylated C-peptide levels above a minimum level of about 0.1 nM betweendosing intervals. In certain aspects, the replacement dose maintains theaverage steady-state concentration C-peptide or PEGylated C-peptidelevels above a minimum level of about 0.2 nM between dosing intervals.In certain aspects, the replacement dose maintains the averagesteady-state concentration C-peptide or PEGylated C-peptide levels abovea minimum level of about 0.4 nM between dosing intervals.

The terms “subcutaneous” or “subcutaneously” or “S.C.” in reference to amode of administration of insulin or PEGylated C-peptide, refers to adrug that is administered as a bolus injection, or via an implantabledevice into the area in, or below the subcutis, the layer of skindirectly below the dermis and epidermis, collectively referred to as thecutis. Preferred sites for subcutaneous administration and/orimplantation include the outer area of the upper arm, just above andbelow the waist, except the area right around the navel (a 2-inchcircle). The upper area of the buttock, just behind the hipbone. Thefront of the thigh, midway to the outer side, 4 inches below the top ofthe thigh to 4 inches above the knee.

The term “single dose” means that the patient has received a single doseof the drug composition or that the repeated single doses have beenadministered with washout periods in between. Unless specificallydesignated as “single dose” or at “steady-state”the pharmacokineticparameters disclosed and claimed herein encompass both single-dose andmultiple-dose conditions.

The term “sequence similarity” refers to the degree of identity orcorrespondence between nucleic acid or amino acid sequences that may ormay not share a common evolutionary origin (see Reeck et al., supra).However, in common usage and in the present application, the term“homologous”, when modified with an adverb such as “highly”, may referto sequence similarity and may or may not relate to a commonevolutionary origin.

By “statistically significant”, it is meant that the result was unlikelyto have occurred by chance. Statistical significance can be determinedby any method known in the art. Commonly used measures of significanceinclude the p-value, which is the frequency or probability with whichthe observed event would occur, if the null hypothesis were true. If theobtained p-value is smaller than the significance level, then the nullhypothesis is rejected. In simple cases, the significance level isdefined at a p-value of 0.05 or less.

As defined herein, the terms “sustained release”, “extended release”, or“depot formulation” refers to the release of a drug such as PEGylatedC-peptide from the sustained release composition or sustained releasedevice which occurs over a period which is longer than that periodduring which the drug would be available following direct I.V. or S.C.administration of a single dose of drug. In one aspect, sustainedrelease will be a release that occurs over a period of at least aboutone to two weeks, about two to four weeks, about one to two months,about two to three months, or about three to six months. In certainaspects, sustained release will be a release that occurs over a periodof about six months to about one year. The continuity of release andlevel of release can be affected by the type of sustained release device(e.g., programmable pump or osmotically-driven pump) or sustainedrelease composition, and type of PEGylated C-peptides used (e.g.,monomer ratios, molecular weight, block composition, and varyingcombinations of polymers), polypeptide loading, and/or selection ofexcipients to produce the desired effect, as more fully describedherein.

Various sustained release profiles can be provided in accordance withany of the methods of the present invention. “Sustained release profile”means a release profile in which less than 50% of the total release ofdrug that occurs over the course of implantation/insertion or othermethod of administering the drug in the body occurs within the first 24hours of administration. In a preferred embodiment of the presentinvention, the extended release profile is selected from the groupconsisting of; a) the 50% release point occurring at a time that isbetween 48 and 72 hours after implantation/insertion or other method ofadministration; b) the 50% release point occurring at a time that isbetween 72 and 96 hours after implantation/insertion or other method ofadministration; c) the 50% release point occurring at a time that isbetween 96 and 110 hours after implantation/insertion or other method ofadministration; d) the 50% release point occurring at a time that isbetween 1 and 2 weeks after implantation/insertion or other method ofadministration; e) the 50% release point occurring at a time that isbetween 2 and 4 weeks after implantation/insertion or other method ofadministration; f) the 50% release point occurring at a time that isbetween 4 and 8 weeks after implantation/insertion or other method ofadministration; g) the 50% release point occurring at a time that isbetween 8 and 16 weeks after implantation/insertion or other method ofadministration; h) the 50% release point occurring at a time that isbetween 16 and 52 weeks (1 year) after implantation/insertion or othermethod of administration; and i) the 50% release point occurring at atime that is between 52 and 104 weeks after implantation/insertion orother method of administration.

Additionally, use of a sustained release composition can reduce the“degree of fluctuation” (“DFL”) of the drugs plasma concentration. DFLis a measurement of how much the plasma levels of a drug vary over thecourse of a dosing interval (C_(max)−C_(m1n)/C_(m1n)). For simple cases,such as I.V. administration, fluctuation is determined by therelationship between the elimination half-life (T_(1/2)) and dosinginterval. If the dosing interval is equal to the half-life then thetrough concentration is exactly half of the peak concentration, and thedegree of fluctuation is 100%. Thus a sustained release composition witha reduced DFL (for the same dosing interval) signifies that thedifference in peak and trough plasma levels has been reduced.Preferably, the patients receiving a sustained release composition ofPEGylated C-peptide have a DFL approximately 50%, 40%, or 30% of the DFLin patients receiving a non-extended release composition with the samedosing interval.

The terms “treating” or “treatment” means to relieve, alleviate, delay,reduce, reverse, improve, manage, or prevent at least one symptom of acondition in a patient. The term “treating” may also mean to arrest,delay the onset (i.e., the period prior to clinical manifestation of adisease), and/or reduce the risk of developing or worsening a condition.

As used herein, the terms “therapeutically effective amount”,“prophylactically effective amount”, or “diagnostically effectiveamount” is the amount of the drug, e.g., insulin or PEGylated C-peptide,needed to elicit the desired biological response followingadministration.

The term “unit-dose forms” refers to physically discrete units suitablefor human and animal patients and packaged individually as is known inthe art. It is contemplated for purposes of the present invention thatdosage forms of the present invention comprising therapeuticallyeffective amounts of drug may include one or more unit doses (e.g.,tablets, capsules, powders, semisolids [e.g., gelcaps or films], liquidsfor oral administration, ampoules or vials for injection, loadedsyringes) to achieve the therapeutic effect. It is further contemplatedfor the purposes of the present invention that a preferred embodiment ofthe dosage form is a subcutaneously injectable dosage form.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviations,per practice in the art. Alternatively, “about” with respect to thecompositions can mean plus or minus a range of up to 20%, preferably upto 10%, more preferably up to 5%.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly indicatesotherwise. Thus, for example, reference to “a molecule” includes one ormore of such molecules, “a reagent” includes one or more of suchdifferent reagents, reference to “an antibody” includes one or more ofsuch different antibodies, and reference to “the method” includesreference to equivalent steps and methods known to those of ordinaryskill in the art that could be modified or substituted for the methodsand pharmaceutical compositions described herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. The following abbreviationslisted in Table A are used in certain sections of the disclosure:

TABLE A LIST OF ABBREVIATIONS ADA Anti-drug antibody AUC Area under thecurve AUC₍₀₋₇₎ Area under the plasma concentration- time curve from timezero to Day 7 AUC₍₀₋₁₄₎ Area under the plasma concentration- time curvefrom time zero to Day 14 AUC_((0-t))/AUC_(tau) Area under the plasmaconcentration- time curve from time zero to the time of the lastquantifiable concentration AUC_((0-inf))/AUC_(inf) Area under the plasmaconcentration- time curve from time zero to infinity Conc. ConcentrationC_(ss) Concentration at steady state CL/F Apparent clearance uncorrectedfor bioavailability (F) CL_(ss)/F Apparent clearance uncorrected forbioavailability (F) at steady state C_(max) Maximum observedconcentration ELISA Enzyme-linked immunosorbent assay F Bioavailabilityor female F_(rel) Relative bioavailability GLP Good Laboratory Practiceh Hours i.v. Intravenous kg Kilogram L Liter M Male mg Milligram mLMilliliter min Minutes MTD maximum tolerated dose ND Not determined ngNanogram NOEL no observed effect level. nM/nmol/L Nanomolar nnolNanomole QC Quality control PEG Polyethylene glycol RIA Radioimmunoassays.c./S.C. Subcutaneous SD Standard deviation T_(1/2) Terminalelimination half-life T_(max) Time to reach C_(max) Vd/F Apparent volumeof distribution following subcutaneous administration, uncorrected forbioavailability (F) Vd_(ss)/F Apparent volume of distribution followingsubcutaneous administration, uncorrected for bioavailability (F) atsteady state wk Week

Although any methods, compositions, reagents, cells, similar orequivalent to those described herein can be used in the practice ortesting of the invention, the preferred methods and materials aredescribed herein.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press;Handbook of Drug Screening, edited by Ramakrishna Seethala, PrabhavathiB. Fernandes (2001, New York, N.Y., Marcel Dekker, ISBN 0-8247-0562-9);Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools forUse at the Bench, edited by Jane Roskams and Linda Rodgers, 2002, ColdSpring Harbor Laboratory, ISBN 0-87969-630-3; Harris, J M, and Zalipsky,S, eds, Poly(ethylene glycol), Chemistry and Biological Applications,ACS, Washington, 1997; Veronese, F., and J. M. Harris, Eds., Peptide andprotein PEGylation, Advanced Drug Delivery Reviews, 54(4) 453-609(2002); Zalipsky, S., et al., “Use of functionalized Poly(EthyleneGlycols) for modification of polypeptides” in Polyethylene GlycolChemistry Biotechnical and Biomedical Applications. Each of thesegeneral texts is herein incorporated by reference.

The publications discussed above are provided solely for theirdisclosure before the filing date of the present application. Nothingherein is to be construed as an admission that the invention is notentitled to antedate such disclosure by virtue of prior invention.

I. Polyethylene Glycol (PEG)

PEG is a well-known polymer with good solubility in many aqueous andorganic solvents, which exhibits low toxicity, lack of immunogenicity,and is clear, colorless, odorless, and stable. For these reasons andothers, PEG has been selected as the preferred polymer for attachment,but it has been employed solely for purposes of illustration and notlimitation. Similar products may be obtained with other water-solublepolymers, including without limitation; polyvinyl alcohol, otherpoly(alkylene oxides) such as poly(propylene glycol) and the like,poly(oxyethylated polyols) such as poly(oxyethylated glycerol) and thelike, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpurrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride, and polyaminoacids. One skilled in the art will be able toselect the desired polymer based on the desired dosage, circulationtime, resistance to proteolysis, and other considerations.

Representative polymeric reagents and methods for conjugating suchpolymers to an active moiety are described in Harris, J. M. andZalipsky, S., Eds, Poly(ethylene glycol), Chemistry and BiologicalApplications, ACS, Washington, 1997; Veronese, F., and J. M. Harris,Eds., Peptide and Protein PEGylation, Advanced Drug Delivery Reviews,54(4); 453-609 (2002); Zalipsky, S., et al., “Use of Functionalized PolyEthylene Glycols) for Modification of Polypeptides” in PolyethyleneGlycol Chemistry: Biotechnical and Biomedical Applications, J. M.Harris, ed., Plenus Press, New York (1992); Zalipsky (1995) AdvancedDrug Reviews 16:157-182; and in Roberts et al., Adv. Drug DeliveryReviews, 54, 459-476 (2002).

A wide variety of PEG derivatives are both commercially available andsuitable for use in the preparation of the PEG-conjugates of theinvention. For example, NOF Corp.'s SUNBRIGHT® Series (www.peg-drug.com)provides numerous PEG derivatives, including methoxypolyethylene glycolsand activated PEG derivatives such as succinimidyl ester, methoxy-PEGamines, maleimides, and carboxylic acids, for coupling by variousmethods to C-peptide and Nektar Therapeutics' Advanced PEGylation alsooffers diverse PEG-coupling technologies to improve the safety andefficacy of therapeutics. Additional PEGs for use in forming a C-peptideconjugate of the invention include those available from Polypure(Norway), from QuantaBioDesign LTD (Ohio) and Sunbio, Inc (South Korea).Further PEG reagents suitable for use in forming a conjugate of theinvention, and methods of conjugation are described in the Pasut. G., etal., Expert Opin. Ther. Patents (2004), 14(6) 859-893.

A search of patents, published patent applications, and relatedpublications will also provide those skilled in the art reading thisdisclosure with significant possible PEG-coupling technologies andPEG-derivatives. For example, U.S. Pat. Nos. 7,026,440; 6,858,736;6,828,401; 6,602,498; 6,495,659; 6,448,369, 6,436,386; 5,990,237;5,932,462; 5,900,461; 5,824,784; 5,739,208; 5,672,662; 5,650,234;5,629,384; 5,252,714; and 4,904,584; the contents of which areincorporated by reference in their entirety, describe such technologiesand derivatives, and methods for their manufacture.

The PEGylated C-peptides according to the invention have PEG moietieswith a molecular weight varying within a range of about 4,000 Da to80,000 Da. The molecular weight ranges will typically be from about 4000Da to about 10,000 Da, from about 10,000 Da to about 20,000 Da, fromabout 20,000 Da to about 30,000 Da, from about 30,000 Da to about 40,000Da, from about 40,000 Da to about 50,000 Da, from about 50,000 Da toabout 60,000 Da, from about 60,000 Da to about 70,000 Da, and from about70,000 Da to about 80,000 Da. Non-limiting examples of average molecularweights of the PEG moieties are about 10,000 Da, about 20,000 Da, about30,000 Da, about 40,000 Da, about 50,000 Da, about 60,000 Da, about70,000 Da, and about 80,000 Da.

Because virtually all PEG polymers exist as mixtures of diverse highmolecular mass, PEG molecular weight (MW) is typically reported asnumber average (M_(n)), weight average (M_(w)), or z-average (M_(z))molecular weights. The weight average is probably the most useful of thethree, because it fairly accounts for the contributions of differentsized chains to the overall behavior of the polymer, and correlates bestwith most of the physical properties of interest.

${{Number}\mspace{14mu} {average}\mspace{14mu} {{MW}({Mn})}} = \frac{\Sigma ({MiNi})}{\Sigma \; {Ni}}$${{Weight}\mspace{14mu} {average}\mspace{14mu} {{MW}({Mw})}} = \frac{\Sigma ( {{Mi}^{2}{Ni}} )}{\Sigma \; ({MiNi})}$${Z\mspace{14mu} {average}\mspace{14mu} {{MW}({Mz})}} = \frac{\Sigma ( {{Mi}^{3}{Ni}} )}{\Sigma \; ( {{Mi}^{2}{Ni}} )}$

where “Ni” is the mole-fraction (or the number-fraction) of moleculeswith molecular weight “Mi” in the polymer mixture. The ratio of Mw to Mnis known as the polydispersity index (PDI), and provides a roughindication of the breadth of the distribution. The PDI approaches 1.0(the lower limit) for special polymers with very narrow MWdistributions.

The PEG groups of the invention will for a given molecular weighttypically consist of a range of ethylene glycol (or ethyleneoxide;OCH₂CH₂) monomers. For example, a PEG group of molecular weight 2000 Dawill typically consist of 43±10 monomers, the average being around 43-44monomers.

The PEG groups of the present invention will typically comprise a numberof subunits, e.g., each n, n₁ or n₂ or n₃ in any of the claimedcompounds may each independently be from about 1 to about 1000, fromabout 1 to about 800, from about 1 to about 600, from about 1 to about400, from about 1 to about 300, from about 1 to about 200. Well-suitedPEG groups are such wherein the number of subunits (i.e. n₁, n₂, and n₃)are independently selected from the group consisting of from about 800to about 1000; from about 800 to about 950; from about 600 to about 850;from about 400 to about 650; from about 200 to about 450, from about 180to about 350; from about 100 to about 150; from about 35 to about 55;from about 42 to about 62; from about 12 to about 25 subunits, fromabout 1 to 10 subunits. In certain embodiments the PEGylated C-peptidewill have a molecular weight of about 40 kDa, and thus n₁ and n₂ foreach PEG chain in the branch chain PEGs will be within the range ofabout 440 to about 550, or about 450 to about 520.

Branched versions of the PEG polymer (e.g., a branched 40,000 Da PEGpolymer comprised of two or more 10,000 Da to 20,000 Da PEG polymers orthe like) having a total molecular weight of any of the foregoing canalso be used.

Representative branched polymers described therein include those havingthe following generalized structure: (PEG)_(y)-[Core]-[Linker];

where “[Core]” is a central or core molecule from which extends 2 ormore PEG arms, the variable “y” represents the number of PEG arms, and“[Linker]” represents an optional linking moiety (as further definedbelow) that typically couples the [Core] to the C-peptide. In onealternative embodiment of the branched chain PEGs, at least one polymerarm possesses a terminal functional group suitable (e.g. NHS moiety) forreaction with C-peptide. Typically the branched chain polymers of theinvention are coupled to the N-terminal amino group of the C-peptide.

In yet further embodiments the linker moiety can represent either ahydrolytically stable, or alternatively, a degradable linker, meaningthat the linkage can be hydrolyzed under physiological conditions, e.g.,the linkage comprises an ester, hydrolysable carbamate, carbonate, orother such group. Hydrolytically degradable linkages, useful not only asa degradable linkage within a polymer backbone, but also, in the case ofcertain embodiments of the invention, for covalently attaching awater-soluble polymer to a C-peptide, include: carbonate; imineresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al. (1997) Polymer Preprints 38(I):582-3); phosphateester, formed, for example, by reacting an alcohol with a phosphategroup; hydrazone, e.g., formed by reaction of a hydrazide and analdehyde; acetal, e.g., formed by reaction of an aldehyde and analcohol; orthoester, formed, for example, by reaction between a formateand an alcohol; and esters, and certain urethane (carbamate) linkages.Illustrative PEG reagents for use in preparing a releasable C-peptideconjugate in accordance with the invention are described in U.S. Pat.Nos. 6,348,558, 5,612,460, 5,840,900, 5,880,131, and 6,376,470.Typically releasable linkers may be attached to any residue inC-peptide, and are not restricted to the N-terminal amino acid.

Branched PEGs such as those represented generally by the formula,(PEG)_(y)-[Core]-[Linker], above can possess 2 polymer arms to about 8polymer arms (i.e., “y” ranges from 2 to about 8). Preferably, suchbranched PEGs typically possess from 2 to about 4 polymer arms,Multi-armed polymers include those having 2, 3, 4, 5, 6, 7 or 8 PEGarms.

Core molecules in branched PEGs as described above include polyols,which are then further functionalized. Such polyols include aliphaticpolyols having from 1 to 10 carbon atoms and from 1 to 10 hydroxylgroups, including ethylene glycol, alkane diols, alkyl glycols,alkylidene alkyl diols, alkyl cycloalkane diols, 1,5-decalindiol,4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidene diols,dihydroxyalkanes, trihydroxyalkanes, and the like. Cycloaliphaticpolyols may also be employed, including straight chained or closed-ringsugars and sugar alcohols, such as mannitol, sorbitol, inositol,xylitol, quebrachitol, threitol, arabitol, erythritol, adonitol,ducitol, facose, ribose, arabinose, xylose, lyxose, rhamnose, galactose,glucose, fructose, sorbose, mannose, pyranose, altrose, talose,tagitose, pyranosides, sucrose, lactose, maltose, and the like.Additional aliphatic polyols include derivatives of glyceraldehyde,glucose, ribose, mannose, galactose, and related stereoisomers. Othercore polyols that may be used include crown ether, cyclodextrins,dextrins and other carbohydrates such as starches and amylose. Typicalpolyols include glycerol, pentaerythritol, sorbitol, andtrimethylolpropane. Other suitable cores include lysine, and otherpolyamines, and PEG moieties comprising multiple functional terminal endgroups.

Illustrative multi-armed PEGs having 2 arms, 3 arms, 4 arms, and 8 armsare known in the art, and are available commercially and/or can beprepared following techniques known to those skilled in the art. (Seegenerally Pasut et al., (2004) Protein, peptide and non-peptide drugPEGylation for therapeutic application Expert Opinin. Ther. Patents14(6) 859-894). Additional branched-PEGs for use in forming a C-peptideconjugate of the present invention include those described in U.S.Patent Application Publication Nos. 20050009988, 20060194940,20090234070, 20070031371, U.S. Pat. Nos. 6,664,331; 6,362,254;6,437,025; 6,541,543; 6,664,331; 6,730,334; 6,774,180; 6,838,528;7,030,278; 7,026,440; 7,053,150; 7,157,546; 7,223,803; 7,265,186;7,419,600; 7,432,330; 7,432,331; 7,511,094; 7,528,202; 7,589,157; andPCT publication numbers WO2005000360, WO2005108463, WO2005107815,WO2005028539 and WO200605108463.

Exemplary linear or multi-armed PEGs for use herein include those ofgeneral formula (I) (II), (III) or (IV) as further described below:

In one aspect, the PEGylated C-peptide comprises a linear PEG of generalformula: (I):

R₁—O—(CH₂CH₂O)_(n1)-[Linker]-[C-peptide]  (I)

wherein;

-   -   R₁=alkyl; and        n₁ is 20 to 800; and wherein the linker is as defined below, and        is attached to the N-terminal amino group of C-peptide.

In another aspect, the PEGylated C-peptide comprises a branched chainPEG of general formula: (II):

wherein;

-   -   R₁=alkyl;    -   n₁ is 20 to 800;    -   n₂ is 20 to 800;    -   wherein each linker is independently defined as below; and    -   wherein the linker connecting to C-peptide is attached to the        N-terminal amino group of C-peptide.

In one embodiment of the PEGylated C-peptides of formula (II), thePEGylated C-peptide has the structure (II A):

wherein;

-   -   R₁=alkyl;    -   n₁ is 20 to 800;    -   n₂ is 20 to 800; and    -   wherein the linker is defined as below, and is attached to the        N-terminal amino group of C-peptide.

In another aspect, the PEGylated C-peptide comprises a branched chainPEG of general formula (III):

wherein;

-   -   R₁=alkyl;    -   n₁ is 20 to 800;    -   n₂ is 20 to 800;    -   wherein each linker is independently defined as below, and    -   wherein the linker connecting to C-peptide is attached to the        N-terminal amino group of C-peptide.

In one embodiment of the PEGylated C-peptides of formula (III), thePEGylated C-peptide has the structure (III A):

wherein;

-   -   R₁=alkyl;    -   n₁ is 20 to 800;    -   n₂ is 20 to 800; and    -   wherein the linker is defined as below, and is attached to the        N-terminal amino group of C-peptide.

In another aspect, the PEGylated C-peptide comprises a branched chainPEG of general formula (IV):

wherein;

-   -   R₁=alkyl;    -   n₁ is 20 to 800;    -   n₂ is 20 to 800;    -   wherein each linker is independently defined as below; and    -   wherein the linker connecting the lysine residue to C-peptide is        attached to the N-terminal amino group of C-peptide and the        C-terminal carboxylate group of the lysine residue, and wherein        the linkers connecting the lysine moiety to the PEG moieties are        linked through the amino groups of the lysine molecule.

In another embodiment of the PEGylated C-peptides of formula (IV), thePEGylated C-peptide has the structure (IV A):

wherein;

-   -   R₁=alkyl;    -   n₁ is 20 to 800;    -   n₂ is 20 to 800; and wherein the linker is defined as below, and        is attached to the N-terminal amino group of C-peptide.

Those of ordinary skill in the art will recognize that the foregoingdiscussion describing linear and branched chain PEGs for use in forminga C-peptide conjugate is by no means exhaustive and is merelyillustrative, and that all polymeric materials, and branched PEGstructures having the qualities described herein are contemplated.Moreover, based on the instant invention, one of ordinary skill in theart can readily determine the appropriate size and optimal structure ofalternative PEGylated C-peptides using routine experimentation, forexample, by obtaining the clearance profile for each conjugate byadministering the conjugate to a patient and taking periodic bloodand/or urine samples, as described herein. Once a series of clearanceprofiles has been obtained for each tested conjugate, a conjugate ormixture of conjugates, having the desired clearance profile(s) can bedetermined.

II. Linker Moieties

The particular linkage between the C-peptide and the water-solublepolymer depends on a number of factors, including the desired stabilityof the linkage, its hydrophobicity, the particular linkage chemistryemployed, and impact on the aqueous solubility, and aggregation state ofthe PEGylated C-peptide. Exemplary linkages are hydrolytically stable,and water soluble, representative suitable linker can comprise anycombination of amide, a urethane (also known as carbamate), amine,thioether (also known as sulfide), or urea (also known as carbamide)groups.

There are many commercially available examples of suitable water-solublelinker moieties and/or these can be prepared following techniques knownto those skilled in the art. Certain illustrative exemplary linkermoieties are described below. The corresponding activated intermediatesare provided in Tables D1 and D2 below.

In one embodiment of the PEGylated C-peptides of general formula (I)(II), (III), or (IV), the PEGylated C-peptide comprises one or morelinkers independently selected from;

-   -   —X—, —CO—, —(CH₂)_(m2)—, —(CH₂)_(m1)—CO—, —CO—(CH₂)_(m1)—,        —CO—X—CO—,    -   —(CH₂)_(m1)—X—(CH₂)_(m1)—, —(CH₂)_(m1)—CO—(CH₂)_(m1)—, —X—CO—X—,        —X—(CH₂)_(m1)—X—,    -   —CO—(CH₂)_(m1)—CO—, —X—CO—(CH₂)_(m1)—, —(CH₂)_(m1)—CO—X—,        —X—(CH₂)_(m1)—CO—X—,    -   —X—CO—(CH₂)_(m1)X—, —X—CO—(CH₂)_(m1)—CO—X—(CH₂)_(m1)—X—CO—,    -   —X—(CH₂)_(m1)—X—CO—(CH₂)_(m2)—,    -   —X—(CH₂)_(m1)—CO—X—(CH₂)_(m2)—,    -   —X—(CH₂)_(m1)—X—CO—(CH₂)_(m2)—X—,    -   —X—(CH₂)_(m1)—X—CO—(CH₂)_(m2)—CO—,    -   —X—(CH₂)_(m1)—CO—X—(CH₂)_(m2)—X—, and    -   —X—(CH₂)_(m1)—CO—X—(CH₂)_(m2)—CO—;

wherein;

-   -   each X is independently selected from —O—, —S—, or —NH— or is        missing;    -   each m₁ is independently 0 to 5; and    -   each m₂ is independently 1 to 5.

In another embodiment of the PEGylated C-peptides of formula (I) (II),(III), or (IV) the PEGylated C-peptide comprises one or more linkersindependently selected from;

-   -   —X₁—(CH₂)_(m4)—CO—;    -   —X₁—CO—;    -   —X₁—CO—(CH₂)_(m4)—CO—;    -   —X₁—CO—X₂—(CH₂)_(m3)—CO—; and    -   X₁—(CH₂)_(m2)—X₂—CO—(CH₂)_(m4)—CO—;

wherein;

-   -   X₁ is —O—, or missing;    -   X₂ is —NH—;    -   m₂ is 1 to 5    -   m₃ is 2; and    -   m₄ is 1 to 5.

In another embodiment of the PEGylated C-peptides of formula (II),(III), or (IV) the PEGylated C-peptide comprises one or more linkersindependently selected from;

-   -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—X—,    -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—(CH₂)_(m5)—CO—,    -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—CO—, and    -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—CO—(CH₂)_(m5)—CO—;

wherein;

-   -   X is independently selected from —O—, —S—, or —NH— or is        missing;    -   X₁ is —O—, or missing;    -   X₂ is —NH—;    -   each m₅ is independently selected from 1 to 5; and    -   each n₃ is independently selected from 1 to 400.

In another embodiment of the PEGylated C-peptides of formula (II), (III)or (IV), the PEGylated C-peptide comprises one or more linkersindependently selected from;

-   -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—(CH₂)_(m6)—CO—,    -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—CO—, and    -   —X₁—CO—X₂—(CH₂)_(m5)—X₁—(CH₂—CH₂—O)_(n3)—CO—(CH₂)_(m7)—CO—;

wherein;

-   -   X₁ is —O—, or is missing;    -   X₂ is —NH—;    -   m₅ is 3;    -   m₆ is independently 2 or 5;    -   m₇ is 3; and    -   n₃ is 1 to 400.

In another embodiment of the PEGylated C-peptides of formula (IV), thePEGylated C-peptide comprises a linker independently selected from;

—X—, —CO—, —(CH₂)_(m2)—, and —X₁—C(O)—X₂—;

wherein;

-   -   X is —O—, or —S—, or —NH— or is missing;    -   X₁ and X₂ are independently selected from —NH—; or —O—, or is        missing; and    -   m₂ is independently 1 to 5.

Those of ordinary skill in the art will recognize that the foregoingdiscussion describing linker moieties for use in forming a C-peptideconjugate is by no means exhaustive and is merely illustrative, and thatall linkers having the qualities described herein are contemplated.

Moreover, based on the teachings described herein, one of ordinary skillin the art can readily determine the appropriate size and optimalstructure of the linker using routine experimentation. For example bytesting a number of different commercially available PEG derivativeswith different linker moieties and characterizing the biologicalactivity, solubility and stability of the resulting PEGylated C-peptide.

III. Activated Functional Groups and Reaction Conditions

The only natural free amino group in human C-peptide is the N-terminalamino group, and thus the selective conjugation of a polymeric PEG groupto the N-terminal amino group of C-peptide can be readily accomplishedusing a variety of commercially available activated PEGs and standardcoupling approaches.

In one approach, a C-peptide is conjugated to the PEG reagent via anactivated functional group, such as an active ester such as asuccinimidyl derivative (e.g., an N-hydroxysuccinimide ester (NHS)). Inthis approach, the PEG bearing the reactive ester is reacted with theC-peptide in aqueous media under appropriate pH conditions, at roomtemperature or 4° C., for a few hours to overnight. Typically thepolymeric reagent is coupled to the activated functional group via alinker as described herein.

N-terminal PEGylation, with a PEG reagent bearing anN-hydroxysuccinimide ester (NHS group), is typically carried out at roomtemperature, or 4° C., in a polar aprotic solvent such asdimethylformamide (DMF) or acetonitrile, or a combination thereof (withsmall amounts of water to solubilize the peptide) under slightly basicpH conditions, e.g., from pHs ranging from about 7.5 to about 8.Reaction times are typically in the range of 1 to 24 hours, dependingupon the pH and temperature of the reaction.

N-terminal PEGylation, with a PEG reagent bearing an aldehyde group, istypically conducted under mild conditions, in the presence of sodiumcyanoborohydride (10 equiv.), 4° C., at pHs from about 5 to 10, forabout 20 to 36 hours. N-terminal pegylation may be conducted, forexample, in 100 mM sodium acetate or 100 mM sodium biphosphate buffer atpH 5.0˜6.0. The buffer may additionally contain 20 mM sodiumcyanoborahydride. The molar ratio of compound to mPEG-aldehyde may be1:5˜1:10. The pegylation is then stirred overnight at ambient orrefrigeration temperature.

N-terminal PEGylation, with a PEG reagent bearingp-Nitrophenyloxycarbonyl group, is typically conducted with borate orphosphate buffer at pHs from about 8 to 8.3, at room temperatureovernight.

For all the coupling reactions, varying ratios of polymeric reagent toC-peptide may be employed, e.g., from an equimolar ratio up to a 10-foldmolar excess of polymer reagent. Typically, up to a 2-fold molar excessof polymer reagent will suffice. Exemplary activated PEGs include, e.g.,those listed in Table D1 and Table D2. In the following list, selectedPEGylation reagents are listed. Obviously other active groups andlinkers may be employed, and are known to those skilled in the art.

TABLE D1 Exemplary Activated Linear PEGs Abbreviation & Molecular WeightRange Structure/Functionality (in Da)

  N-hydroxysuccinimide ester SUNBRIGHT ME-020CS MW = 2,000 SUNBRIGHTME-050CS MW = 5,000 SUNBRIGHT ME-100CS MW = 10,000 SUNBRIGHT ME-200CS MW= 20,000 SUNBRIGHT ME-300CS MW = 30,000 SUNBRIGHT ME-400CS MW = 40,000

  N-hydroxysuccinimide ester SUNBRIGHT ME-050GS MW = 5,000 SUNBRIGHTME-200GS MW = 20,000 SUNBRIGHT ME-300GS MW = 30,000 SUNBRIGHT ME-400GSMW = 40,000

  N-hydroxysuccinimide ester SUNBRIGHT ME-050TS MW = 5,000 SUNBRIGHTME-200TS MW = 20,000 SUNBRIGHT ME-300TS MW = 30,000 SUNBRIGHT ME-400TSMW = 40,000

  N-hydroxysuccinimide ester SUNBRIGHT ME-020AS MW = 2,000 SUNBRIGHTME-050AS MW = 5,000

  N-hydroxysuccinimide ester SUNBRIGHT ME-050HS MW = 5,000 SUNBRIGHTME-200HS MW = 20,000 SUNBRIGHT ME-300HS MW = 30,000 SUNBRIGHT ME-400HSMW = 40,000

  p-Nitrophenyl SUNBRIGHT MENP-020H MW = 2,000 SUNBRIGHT MENP-050H MW =5,000 SUNBRIGHT MENP-10T MW = 10,000 SUNBRIGHT MENP-20T MW = 20,000SUNBRIGHT MENP-30T MW = 30,000 SUNBRIGHT MENP-40T MW = 40,000CH₃—O—(CH₂CH₂O)_(n1)—N═C═O Isocyanate

  Aldehyde SUNBRIGHT ME-050AL MW = 5,000 SUNBRIGHT ME-100AL MW = 10,000SUNBRIGHT ME-200AL MW = 20,000 SUNBRIGHT ME-300AL MW = 30,000 SUNBRIGHTME-400AL MW = 40,000

  Aldehyde SUNBIO P1PAL-5 MW = 5,000 SUNBIO P1PAL-10 MW = 10,000 SUNBIOP1PAL-20 MW = 20,000 SUNBIO P1PAL-30 MW = 30,000

  Amide Aldehyde SUNBIO P1APAL-5 MW = 5,000 SUNBIO P1APAL-10 MW = 10,000SUNBIO P1APAL-20 MW = 20,000 SUNBIO P1APAL-30 MW = 30,000

  Urethane Aldehyde SUNBIO P1TPAL-5 MW = 5,000

  Aldehyde SUNBIO P1BAL-5 MW = 5,000 SUNBIO P1BAL-10 MW = 10,000 SUNBIOP1BAL-20 MW = 20,000 SUNBIO P1BAL-30 MW = 30,000

  Amide Aldehyde SUNBIO P1ABAL-5 MW = 5,000 SUNBIO P1ABAL-10 MW = 10,000SUNBIO P1ABAL-20 MW = 20,000 SUNBIO P1ABAL-30 MW = 30,000

  Urethane Aldehyde SUNBIO P1TBAL-5 MW = 5,000

  N-hydroxysuccinimide ester X = 0, y = 1 SUNBRIGHT-AS X = 0, y = 5SUNBRIGHT-HS X = 1, y = 2 SUNBRIGHT-CS X = 1, y = 3 SUNBRIGHT-GS

  Maleimide z = 2 SUNBRIGHT-MA z = 5 SUNBRIGHT-MA3

TABLE D2 Exemplary Activated Branched PEGs Abbreviation & MolecularStructure/Functionality Weight Range (in Da)

  N-hydroxysuccinimide ester SUNBRIGHT GL2-200GS2 MW = 20,000 SUNBRIGHTGL2-400GS2 MW = 40,000 SUNBRIGHT GL2-400GS2 MW = 60,000 SUNBRIGHTGL2-800GS2 MW = 80,000

  p-Nitrophenyl SUNBRIGHT GL2-100NP MW = 10,000 SUNBRIGHT GL2-200NP MW =20,000 SUNBRIGHT GL2-400NP MW = 40,000 SUNBRIGHT GL2-600NP MW = 60,000SUNBRIGHT GL2-800NP MW = 80,000

  N-hydroxysuccinimide ester SUNBRIGHT GL2-200TS MW = 20,000 SUNBRIGHTGL2-400TS MW = 40,000 SUNBRIGHT GL2-600TS MW = 60,000 SUNBRIGHTGL2-800TS MW = 80,000

  Aldehyde SUNBRIGHT GL2-200AL3 MW = 20,000 SUNBRIGHT GL2-400AL3 MW =40,000 SUNBRIGHT GL2-600AL3 MW = 60,000 SUNBRIGHT GL2-800AL3 MW = 80,000

  Aldehyde SUNBRIGHT GL3-400AL100U MW = 50,000

  p-Nitrophenyl SUNBRIGHT GL3-400NP100U MW = 50,000

  N-hydroxysuccinimide ester SUNBRIGHT GL3-400GS100U MW = 50,000

  N-hydroxysuccinimide ester SUNBRIGHT GL3-400HS100U MW = 50,000

  N-hydroxysuccinimide ester SUNBRIGHT LY-400NS MW = 40,000

MW = 40,000

The PEGylated C-peptide can be purified after neutralization of thereaction buffer, by any convenient approach, e.g., by precipitation withisopropyl-ether followed by reverse phase HPLC or ion exchangechromatography.

IV. Therapeutic Forms of C-Peptide

The terms “C-peptide” or “proinsulin C-peptide” as used herein includesall naturally-occurring and synthetic forms of C-peptide that retainC-peptide activity. Such C-peptides include the human peptide, as wellas peptides derived from other animal species and genera, preferablymammals. Preferably, “C-peptide” refers to human C-peptide having theamino acid sequence EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ. ID. No. 1 inTable D3).

C-peptides from a number of different species have been sequenced, andare known in the art to be at least partially functionallyinterchangeable. It would thus be a routine matter to select a variantbeing a C-peptide from a species or genus other than human. Several suchvariants of C-peptide (i.e., representative C-peptides from otherspecies) are shown in Table D3 (see SEQ. ID. Nos. 1-29).

Table D3 C-peptide Variants human Human EAEDLQVGQVELGGGPGAGSLQPLALEGSLQgb|AAA72531.1| M-proinsulin (SEQ. ID. No. 1) dbj|BAH59081.1| Pan(SEQ. ID. No. 1) EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ NP_001008996.1|troglodytes Alignment EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ emb|CAA43403.1|(SEQ. ID. No. 2) EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ GENE ID: 449570Identities = 31/31 (100%), Positives = 31/31 (100%), INS Gaps =0/31 (0%) Gorilla (SEQ. ID. No. 1) EAEDLQVGQVELGGGPGAGSLQPLALEGSLQgb|AAN06935.1| gorilla Alignment EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ(SEQ. ID. No. 3) EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ Identities =31/31 (100%), Positives = 31/31 (100%), Gaps = 0/31 (0%) Pongo(SEQ. ID. No. 1) EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ gb|AAN06937.1| pygmaeusEAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (Bornean (SEQ. ID. No. 4)EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ orangutan) Identities =31/31 (100%), Positives = 31/31 (100%), Gaps = 0/31 (0%) Chlorocebus(SEQ. ID. No. 1) EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ emb|CAA43405.1|aethiops EAED QVGQVELGGGPGAGSLQPLALEGSLQ (Monkey) (SEQ. ID. No. 5)EAEDPQVGQVELGGGPGAGSLQPLALEGSLQ Identities = 30/31 (96%), Positives =30/31 (96%), Gaps = 0/31 (0%) Canis lupus (SEQ. ID. No. 1)EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ ref|NP_001|23565.1| familiarisE EDLQV  VEL G PG G LQPLALEG+LQ sp|P01321.1| (Dog) (SEQ. ID. No. 6)EVEDLQVRDVELAGAPGEGGLQPLALEGALQ INS_CANFAemb| Identities =23/31 (74%), Positives = 24/31 (77%), CAA23475.1| Gaps = 0/31 (0%)GENE ID: 483665  INS Oryctolagus (SEQ. ID. No. 1)EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ gb|ACK44319.1| cuniculusE E+LQVGQ ELGGGP AG LQP ALE +LQ (Rabbit) (SEQ. ID. No. 7)EVEELQVGQAELGGGPDAGGLQPSALELALQ Identities = 23/31 (74%), Positives =25/31 (80%), Gaps = 0/31 (0%) Rattus (SEQ. ID. No. 1)EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ ref1NP 062003.1| norvegicusE ED QV Q+ELGGGPGAG LQ LALE + Q sp|P01323.1| (SEQ. ID. No. 8)EVEDPQVAQLELGGGPGAGDLQTLALEVARQ INS2_RAT Identities =22/31 (70%), Positives = 24/31 (77%), emb|CAA24560.1| Gaps = 0/31 (0%) GENE ID:24506 Ins2 Apodemus (SEQ. ID. No. 1)EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ gb|ABB89748.1| semotusE ED QV Q+ELGGGPGAG LQ LALE + Q (Taiwan (SEQ. ID. No. 9)EVEDPQVAQLELGGGPGAGDLQTLALEVARQ field Identities =22/31 (70%), Positives = 24/31 (77%), mouse) Gaps = 0/31 (0%) Geodia(SEQ. ID. No. 1) EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ pir||S09278 cydoniumE ED QVGQVELG GPGAGS Q LALE + Q sponge (SEQ. ID. No. 10)EVEDPQVGQVELGAGPGAGSEQTLALEVARQ Identities = 23/31 (74%), Positives =24/31 (77%), Gaps = 0/31 (0%) Mus musculus (SEQ. ID. No. 1)EAEDLQVGQVELGGGPGAGSLQPLALE ref|NP_032413.1| E ED QV Q+ELGGGPGAG LQ LALEsp|P01326.1| (SEQ. ID. No. 11) EVEDPQVAQLELGGGPGAGDLQTLALEINS2_MOUSEemb| Identities = 21/27 (77%), Positives = 22/27 (81%),CAA28433.1| Gaps = 0/27 (0%) GENE ID: 16334 Ins2 Mus caroli(SEQ. ID. No. 1) EAEDLQVGQVELGGGPGAGSLQPLALE gb|ABB89749.1| (RyukyuE ED QV Q+ELGGGPGAG LQ LALE mouse) (SEQ. ID. No. 12)EVEDPQVAQLELGGGPGAGDLQTLALE Identities = 21/27 (77%), Positives =22/27 (81%), Gaps = 0/27 (0%) Rattus (SEQ. ID. No. 1)EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ prf||720460B norvegicusE ED QV Q+ELGGGPGAG LQ LALE + Q (SEQ. ID. No. 13)EVEDPQVPQLELGGGPGAGDLQTLALEVARQ Identities = 22/31 (70%), Positives =24/31 (77%), Gaps = 0/31 (0%) Rattus losea (SEQ. ID. No. 1)EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ gb|ABB89747.1|E ED QV Q ELGGGPGAG LQ LALE + Q (SEQ. ID. No. 14)EVEDPQVAQQELGGGPGAGDLQTLALEVARQ Identities = 22/31 (70%), Positives =23/31 (74%), Gaps = 0/31 (0%) Niviventer  (SEQ. ID. No. 1)EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ gb|ABB89750.1| coxingiE ED QV Q+ELGGGPG G LQ LALE + Q (Coxing's (SEQ. ID. No. 15)EVEDPQVPQLELGGGPGTGDLQTLALEVARQ white- Identities =21/31 (67%), Positives = 23/31 (74%), bellied rat) Gaps = 0/31 (0%)Microtus (SEQ. ID. No. 1) AEDLQVGQVELGGGPGAGSLQPLALE gb|ABB89752.1|kikuchii  ED QV Q+ELGGGPGAG LQ LALE (Taiwan (SEQ. ID. No. 16)VEDPQVAQLELGGGPGAGDLQTLALE vole) Identities = 20/26 (76%), Positives =21/26 (80%), Gaps = 0/26 (0%) Rattus (SEQ. ID. No. 1)EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ ref|NP_062002.1| norvegicusE ED QV Q+ELGGGP AG LQ LALE + Q gb|AAA41439.1| Insulin 1(SEQ. ID. No. 17) EVEDPQVPQLELGGGPEAGDLQTLALEVARQ gb|AAA41442.1|precursor Identities = 21/31 (67%), Positives = 23/31 (74%),emb|CAA24559.1| Gaps = 0/31 (0%) gb|EDL94407.1| GENE ID: 24505  Ins1Felis catus (SEQ. ID. No. 1) EAEDLQVGQVELGGGPGAGSLQPLALEGSLQref|NP_001009272.1| (Domestic EAEDLQ    ELGPGAG   LQP ALE  LQsp|P06306.2| cat) (SEQ. ID. No. 18) EAEDLQGKDAELGEAPGAGGLQPSALEAPLQINS_FELCA Identities = 21/31 (67%), Positives = 21/31 (67%),dbj|BAB84110.1| Gaps = 0/31 (0%) GENE ID: 493804 INS Golden(SEQ. ID. No. 1) AEDLQVGQVELGGGPGAGSLQPLALE sp|P01313.2| hamster ED QV Q+ELGGGPGAL  Q LALE INS_CRILO (SEQ. ID. No. 19)VEDPQVAQLELGGGPGADDLQTLALE pir||I48166 Identities =19/26 (73%), Positives = 20/26 (76%), gb|AAA37089.1| Gaps = 0/26 (0%)Niviventer (SEQ. ID. No. 1) EAEDLQVGQVELGGGPGAGSLQPLALEGSLQgb|ABB89746.1| coxingi E ED QV Q+ELG GP AG LQ LALE + Q (Coxing's(SEQ. ID. No. 20)  EVEDPQVAQLELGEGPEAGDLQTLALEVARQ white- Identities =20/31 (64%), Positives = 22/31 (70%), bellied rat) Gaps = 0/31 (0%)Apodemus (SEQ. ID. No.1) EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ gb|ABB89744.1|semotus E ED QV Q+ELGG PG G L+ LALE + Q (Taiwan (SEQ. ID. No.21)EVEDPQVEQLELGGAPGTGDLETLALEVARQ field Identities =19/31 (61%), Positives = 22/31 (70%), mouse) Gaps = 0/31 (0%)Rattus losea (SEQ. ID. No. 1) EAEDLQVGQVELGGGPGAGSLQPLALEGSLQgb|ABB89743.1| E ED QV Q+ELGG P AG LQ LALE + Q (SEQ. ID. No. 22)EVEDPQVPQLELGGSPEAGDLQTLALEVARQ Identities = 20/31 (64%), Positives =22/31 (70%), Gaps = 0/31 (0%) Meriones (SEQ. ID. No. 1)AEDLQVGQVELGGGPGAGSLQPLALEGSLQ gb|ABB89751.1| unguiculatus  ED Q+Q+ELGG PGAG LQ LALE + Q (Mongolian (SEQ. ID. No. 23)VEDPQMPQLELGGSPGAGDLQALALEVARQ gerbil) Identities =19/30 (63%), Positives = 22/30 (73%), Gaps = 0/30 (0%) Psammomys(SEQ. ID. No. 1) AEDLQVGQVELGGGPGAGSLQPLALEGSLQ sp|Q62587.1| obesus +D Q+ Q+ELGG PGAG L+ LALE + Q INS_PSAOB (Fat sand (SEQ. ID. No. 24)VDDPQMPQLELGGSPGAGDLRALALEVARQ emb|CAA66897.1| rat) Identities =17/30 (56%), Positives = 22/30 (73%), Gaps = 0/30 (0%) Sus scrofa(SEQ. ID. No. 1) EAEDLQVGQVELGGGPGAGSLQPLALEG ref|NP_001103242.1| (Pig)EAE+ Q G VELGG  G G LQ LALEG (SEQ. ID. No. 25)EAENPQAGAVELGG--GLGGLQALALEG Identities = 19/28 (67%), Positives =20/28 (71%), Gaps = 2/28 (7%) Rhinolophus (SEQ. ID. No. 26)gb|ACC68945.1| ferrumequinum EVEDPQAGQVELGGGPGTGGLQSLALEGPPQ Equus(SEQ. ID. No. 27) GENE ID: 100060077 przewalskiiEAEDPQVGEVELGGGPGLGGLQPLALAGPQQ LOC100060077 (Horse) gb|AAB25818.1|Bos Taurus (SEQ. ID. No. 28) gb|AAI42035.1| (Bovine)EVEGPQVGALELAGGPGAGGLEGPPQ Otolemur (SEQ. ID. No. 29) gb|ACH53103.1|garnettii DTEDPQVGQVGLGGSPITGDLQSLALDVPPQ (Small-eared galago)

Thus all such homologues, orthologs, and naturally-occurring isoforms ofC-peptide from human as well as other species (SEQ. ID Nos. 1-29) areincluded in any of the methods and pharmaceutical compositions of theinvention, as long as they retain detectable C-peptide activity.

The C-peptides may be in their native form, i.e., as different variantsas they appear in nature in different species which may be viewed asfunctionally equivalent variants of human C-peptide, or they may befunctionally equivalent natural derivatives thereof, which may differ intheir amino acid sequence, e.g., by truncation (e.g., from the N- orC-terminus or both) or other amino acid deletions, additions,insertions, substitutions, or post-translational modifications.Naturally-occurring chemical derivatives, including post-translationalmodifications and degradation products of C-peptide, are alsospecifically included in any of the methods and pharmaceuticalcompositions of the invention including, e.g., pyroglutamyl,iso-aspartyl, proteolytic, phosphorylated, glycosylated, oxidatized,isomerized, and deaminated variants of C-peptide.

It is known in the art to synthetically modify the sequences of proteinsor peptides, while retaining their useful activity, and this may beachieved using techniques which are standard in the art and widelydescribed in the literature, e.g., random or site-directed mutagenesis,cleavage, and ligation of nucleic acids, or via the chemical synthesisor modification of amino acids or polypeptide chains. Similarly it iswithin the skill in the art to address and/or mitigate immunogenicityconcerns if they arise using C-peptide variants, e.g., by the use ofautomated computer recognition programs to identify potential T cellepitopes, and directed evolution approaches to identify less immunogenicforms.

Any such modifications, or combinations thereof, may be made and used inany of the methods and pharmaceutical compositions of the invention, aslong as activity is retained. The C-terminal end of the molecule isknown to be important for activity. Preferably, therefore, theC-terminal end of the C-peptide should be preserved in any suchC-peptide variants or derivatives, more preferably the C-terminalpentapeptide of C-peptide (EGSLQ) (SEQ. ID. No. 31) should be preservedor sufficient (see Henriksson M et al.: Cell Mol. Life. Sci. 62:1772-1778, (2005)). As mentioned above, modification of an amino acidsequence may be by amino acid substitution, e.g., an amino acid may bereplaced by another that preserves the physicochemical character of thepeptide (e.g., A may be replaced by G or vice versa, V by A or L; E by Dor vice versa; and Q by N). Generally, the substituting amino acid hassimilar properties, e.g., hydrophobicity, hydrophilicity,electronegativity, bulky side chains, etc., to the amino acid beingreplaced.

Modifications to the mid-part of the C-peptide sequence (e.g., toresidues 13 to 25 of human C-peptide) allow the production of functionalderivatives or variants of C-peptide. Thus, C-peptides which may be usedin any of the methods or pharmaceutical compositions of the inventionmay have amino acid sequences which are substantially homologous, orsubstantially similar to the native C-peptide amino acid sequences,e.g., to the human C-peptide sequence of SEQ. ID. No. 1 or any of theother native C-peptide sequences shown in Table D3. Alternatively, theC-peptide may have an amino acid sequence having at least 30% preferablyat least 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% identity withthe amino acid sequence of any one of SEQ. ID. Nos. 1-29 as shown inTable D3, preferably with the native human sequence of SEQ. ID. No. 1.In a preferred embodiment, the C-peptide for use in any of the methodsor pharmaceutical compositions of the present invention is at least 80%identical to a sequence selected from Table D3. In another aspect, theC-peptide for use in any of the methods or pharmaceutical compositionsof the invention is at least 80% identical to human C-peptide (SEQ. ID.No. 1). Although any amino acid of C-peptide may be altered as describedabove, it is preferred that one or more of the glutamic acid residues atpositions 3, 11, and 27 of human C-peptide (SEQ. ID. No. 1) orcorresponding or equivalent positions in C-peptide of other species, areconserved. Preferably, all of the glutamic acid residues at positions 3,11, and 27 (or corresponding Glu residues) of SEQ. ID. No. 1 areconserved. Alternatively, it is preferred that Glu27 of human C-peptide(or a corresponding Glu residue of a non-human C-peptide) is conserved.An exemplary functional equivalent form of C-peptide which may be usedin any of the methods or pharmaceutical compositions of the inventionincludes the amino acid sequences:

(SEQ. ID. No. 30) EXEXXQXXXXELXXXXXXXXXXXXALBXXXQ. (SEQ. ID. No. 33)GXEXXQXXXXELXXXXXXXXXXXXALBXXXQ.

As used herein, X is any amino acid. The N-terminal residue may beeither Glu or Gly (SEQ. ID. No. 30 or SEQ. ID. No. 33, respectively).Functionally equivalent derivatives or variants of native C-peptidesequences may readily be prepared according to techniques well-known inthe art, and include peptide sequences having a functional, e.g., abiological activity of a native C-peptide.

Fragments of native or synthetic C-peptide sequences may also have thedesirable functional properties of the peptide from which they werederived and may be used in any of the methods or pharmaceuticalcompositions of the invention. The term “fragment” as used herein thusincludes fragments of a C-peptide provided that the fragment retains thebiological or therapeutically beneficial activity of the whole molecule.The fragment may also include a C-terminal fragment of C-peptide.Preferred fragments comprise residues 15-31 of native C-peptide, moreespecially residues 20-31. Peptides comprising the pentapeptide EGSLQ(SEQ. ID. No. 31) (residues 27-31 of native human C-peptide) are alsopreferred. The fragment may thus vary in size from, e.g., 4 to 30 aminoacids or 5 to 20 residues. Suitable fragments are disclosed in WO98/13384 the contents of which are incorporated herein by reference.

The fragment may also include an N-terminal fragment of C-peptide,typically having the sequence EAEDLQVGQVEL (SEQ. ID. No. 32), or afragment thereof which comprises 2 acidic amino acid residues, capableof adopting a conformation where said two acidic amino acid residues arespatially separated by a distance of 9-14 A between the alpha-carbonsthereof. Also included are fragments having N- and/or C-terminalextensions or flanking sequences. The length of such extended peptidesmay vary, but typically are not more than 50, 30, 25, or 20 amino acidsin length. Representative suitable fragments are described in U.S. Pat.No. 6,610,649, which is hereby incorporated by reference in itsentirety.

In such a case it will be appreciated that the extension or flankingsequence will be a sequence of amino acids which is not native to anaturally-occurring or native C-peptide, and in particular a C-peptidefrom which the fragment is derived. Such a N- and/or C-terminalextension or flanking sequence may comprise, e.g., from 1 to 10, 1 to 6,1 to 5, 1 to 4, or 1 to 3 amino acids.

The term “derivative” as used herein thus refers to C-peptide sequencesor fragments thereof, which have modifications as compared to the nativesequence. Such modifications may be one or more amino acid deletions,additions, insertions, and/or substitutions. These may be contiguous ornon-contiguous. Representative variants may include those having 1 to 6,or more preferably 1 to 4, 1 to 3, or 1 or 2 amino acid substitutions,insertions, and/or deletions as compared to any of SEQ. ID. Nos. 1-33.The substituted amino acid may be any amino acid, particularly one ofthe well-known 20 conventional amino acids (Ala (A); Cys (C); Asp (D);Glu (E); Phe (F); Gly (G); His (H); Ile (I); Lys (K); Leu (L); Met (M);Asn (N); Pro (P); Gin (Q); Arg (R); Ser (S); Thr (T); Val (V); Trp (W);and Tyr (Y)). Any such variant or derivative of C-peptide may be used inany of the methods or pharmaceutical compositions of the invention.

Isomers of the native L-amino acids, e.g., D-amino acids may beincorporated in any of the above forms of C-peptide, and used in any ofthe methods or pharmaceutical compositions of the invention. Additionalvariants may include amino and/or carboxyl terminal fusions as well asintrasequence insertions of single or multiple amino acids. Longerpeptides may comprise multiple copies of one or more of the C-peptidesequences, such as any of SEQ. ID. Nos. 1-33. Insertional amino acidsequence variants are those in which one or more amino acid residues areintroduced at a site in the protein. Deletional variants arecharacterized by the removal of one or more amino acids from thesequence. Variants may include, e.g., different allelic variants as theyappear in nature, e.g., in other species or due to geographicalvariation. All such variants, derivatives, fusion proteins, or fragmentsof C-peptide are included, may be used in any of the methods claims orpharmaceutical compositions disclosed herein, and are subsumed under theterm “C-peptide”.

The PEGylated forms of C-peptide, C-peptide variants, derivatives, andfragments thereof are functionally equivalent in that they havedetectable C-peptide activity. More particularly, they exhibit at leastabout 1%, at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 100%, or higher than 100% of theactivity of native proinsulin C-peptide, particularly human C-peptide.Thus, they are capable of functioning as proinsulin C-peptide, i.e., cansubstitute for C-peptide itself. Such activity means any activityexhibited by a native C-peptide, whether a physiological responseexhibited in an in vivo or in vitro test system, or any biologicalactivity or reaction mediated by a native C-peptide, e.g., in an enzymeassay or in binding to test tissues, membranes, or metal ions. Thus, itis known that C-peptide causes an influx of calcium and initiates arange of intracellular signalling cascades such as phosphorylation ofthe MAP-kinase pathway including phosphorylation of ERK 1 and 2, CREB,PKC, GSK3, PI3K, NF-kappaB, and PPARgamma, resulting in an increasedexpression of eNOS, Na+K+ATPase and a wide range of transcriptionfactors. An assay for C-peptide activity can thus be made by assayingfor the activation or up-regulation of any of these pathways uponaddition or administration of the peptide (e.g., fragment or derivative)in question to cells from relevant target tissues including endothelial,kidney, fibroblast and immune cells. Such assays are described in, e.g.,Ohtomo Y et al. (Diabetologia 39: 199-205, (1996)), Kunt T et al.(Diabetologia 42(4): 465-471, (1999)), Shafqat J et al. (Cell Mol. Life.Sci. 59: 1185-1189, (2002)). Kitamura T et al. (Biochem. J. 355:123-129, (2001)), Hills and Brunskill (Exp Diab Res 2008), as describedin WO 98/13384 or in Ohtomo Y et al. (supra) or Ohtomo Y et al.(Diabetologia 41: 287-291, (1998)). An assay for C-peptide activitybased on endothelial nitric oxide synthase (eNOS) activity is alsodescribed in Kunt T et al. (supra) using bovine aortic cells and areporter cell assay. Binding to particular cells may also be used toassess or assay for C-peptide activity, e.g., to cell membranes fromhuman renal tubular cells, skin fibroblasts, and saphenous veinendothelial cells using fluorescence correlation spectroscopy, asdescribed, e.g., in Rigler R et al. (PNAS USA 96: 13318-13323, (1999)),Henriksson M et al. (Cell Mol. Life Sci. 57: 337-342, (2000)) andPramanik A et al. (Biochem Biophys. Res. Commun. 284: 94-98, (2001)).

In another aspect of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 5-fold greater than unmodified C-peptide when subcutaneouslyadministered to a mammal.

In another aspect of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 6-fold greater than unmodified C-peptide when subcutaneouslyadministered to a mammal.

In another aspect of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 7-fold greater than unmodified C-peptide when subcutaneouslyadministered to a mammal.

In another aspect of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 8-fold greater than unmodified C-peptide when subcutaneouslyadministered to a mammal.

In another aspect of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 10-fold greater than unmodified C-peptide whensubcutaneously administered to a mammal.

In another aspect of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 15-fold greater than unmodified C-peptide whensubcutaneously administered to a mammal.

In another aspect of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 20-fold greater than unmodified C-peptide whensubcutaneously administered to a mammal.

In another aspect of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 25-fold greater than unmodified C-peptide whensubcutaneously administered to a mammal.

In another aspect of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 50-fold greater than unmodified C-peptide whensubcutaneously administered to a mammal.

In another aspect of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 75-fold greater than unmodified C-peptide whensubcutaneously administered to a mammal.

In another aspect of any of the claimed PEGylated C-peptides, thePEGylated C-peptide has a plasma or sera pharmacokinetic AUC profile atleast about 100-fold greater than unmodified C-peptide whensubcutaneously administered to a mammal.

In one aspect the mammal is a dog. In one aspect the mammal is a rat. Inone aspect the mammal is a human.

V. C-Peptide and PEGylated C-Peptide Production

C-peptide may be produced synthetically using standard solid-phasepeptide synthesis, or by recombinant technology, e.g., as a by-productin the production of human insulin from human proinsulin, or usinggenetically modified host (see generally WO 1999007735; Jonasson P, etal., J Biotechnol. (2000) 76(2-3):215-26; Jonasson P, et al., Gene(1998);210(2):203-10; Li S X, Tian et al., Sheng Wu Hua Xue Yu Sheng WuWu Li Xue Bao (Shanghai) (2003) 35(11):986-92; Nilsson J, et al., JBiotechnol. (1996) 48(3):241-50; Huang Y B, et al., Acta Biochim BiophysSin (Shanghai) (2006) 38(8):586-92).

In an alternative approach to direct coupling to the N-terminus, the PEGreagent, or a lysine residue, may be incorporated at a desired positionof the C-peptide during peptide synthesis. In this way, site-selectiveintroduction of one or more PEGs can be achieved. See, e.g.,International Patent Publication No. WO 95/00162, which describes thesite selective synthesis of conjugated peptides.

C-peptide can be produced by expressing a DNA sequence encoding theC-peptide in question in a suitable host cell by well known techniquesused for insulin biosynthesis as disclosed in, e.g., U.S. Pat. No.6,500,645. The C-peptide may be expressed directly, or as a multimerizedconstruct to increase the yield of product as disclosed in U.S. Pat. No.6,558,924. The multimerized product is cleaved in vitro after isolationfrom the culture broth.

The polynucleotide sequence coding for the C-peptide may be preparedsynthetically by established standard methods, e.g., the phosphoamiditemethod described by Beaucage et al. (1981) Tetrahedron Letters22:1859-1869, or the method described by Matthes et al. (1984) EMBOJournal 3:801-805. According to the phosphoramidite method,oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer,purified, duplexed and ligated to form the synthetic DNA construct. Acurrently preferred way of preparing the DNA construct is by polymerasechain reaction (PCR).

The polynucleotide sequences may also be of mixed genomic, cDNA, andsynthetic origin. For example, a genomic or cDNA sequence encoding aleader peptide may be joined to a genomic or cDNA sequence encoding theA and B chains, after which the DNA sequence may be modified at a siteby inserting synthetic oligonucleotides encoding the desired amino acidsequence for homologous recombination in accordance with well-knownprocedures or preferably generating the desired sequence by PCR usingsuitable oligonucleotides.

The recombinant method will typically make use of a vector which iscapable of replicating in the selected microorganism or host cell andwhich carries a polynucleotide sequence encoding the parent single-chaininsulin of the invention. The recombinant vector may be an autonomouslyreplicating vector, i.e., a vector which exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid, an extra-chromosomal element, amini-chromosome, or an artificial chromosome.

The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used. The vector may be linear orclosed circular plasmids and will preferably contain an element(s) thatpermits stable integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

The recombinant expression vector is capable of replicating in yeast.Examples of sequences which enable the vector to replicate in yeast arethe yeast plasmid 2 pm replication genes REP 1-3 and origin ofreplication. The vector may contain one or more selectable markers whichpermit easy selection of transformed cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototroph to auxotroph, and the like.Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol ortetracycline resistance. Selectable markers for use in a filamentousfungal host cell include amdS (acetamidase), argB (ornithinecarbamoyltransferase), pyrG (orotidine-5′-phosphate decarboxylase) andtrpC (anthranilate synthase. Suitable markers for yeast host cells areADE2, H153, LEU2, LYS2, MET3, TRP1, and URA3. A well-suited selectablemarker for yeast is the Schizosaccharomyces pompe TPI gene (Russell(1985) Gene 40:125-130).

In the vector, the polynucleotide sequence is operably connected to asuitable promoter sequence. The promoter may be any nucleic acidsequence which shows transcriptional activity in the host cell of choiceincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extra-cellular or intracellular polypeptides eitherhomologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription in abacterial host cell are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and Bacilluslicheniformis penicillinase gene (penP). Examples of suitable promotersfor directing the transcription in a filamentous fungal host cell arepromoters obtained from the genes for Aspergillus oryzae TAKA amylase,Rhizomucor miehei aspartic proteinase, Aspergillus niger neutralalpha-amylase, and Aspergillus niger acid stable alpha-amylase. In ayeast host, useful promoters are the Saccharomyces cerevisiae Mal, TPI,ADH, or PGK promoters. The polynucleotide sequence encoding theC-peptide of the invention will also typically be operably connected toa suitable terminator. In yeast a suitable terminator is the TPIterminator (Alber et al. (1982) J. Mol. Appl. Genet. 1:419-434).

The procedures used to ligate the polynucleotide sequence encoding theparent single-chain insulin of the invention, the promoter and theterminator, respectively, and to insert them into a suitable vectorcontaining the information necessary for replication in the selectedhost, are well known to persons skilled in the art. It will beunderstood that the vector may be constructed either by first preparinga DNA construct containing the entire DNA sequence encoding thesingle-chain insulins of the invention, and subsequently inserting thisfragment into a suitable expression vector, or by sequentially insertingDNA fragments encoding genetic information for the individual elementsfollowed by ligation.

The vector comprising the polynucleotide sequence encoding the C-peptideof the invention is introduced into a host cell so that the vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication. The host cell may be aunicellular microorganism, e.g., a prokaryote, or a non-unicellularmicroorganism, e.g., a eukaryote. Useful unicellular cells are bacterialcells such as gram positive bacteria including, but not limited to, aBacillus cell, Streptomyces cell, or gram negative bacteria such as E.coli and Pseudomonas sp. Eukaryote cells may be mammalian, insect,plant, or fungal cells. In one embodiment, the host cell is a yeastcell. The yeast organism may be any suitable yeast organism which, oncultivation, produces large amounts of the single chain insulin of theinvention. Examples of suitable yeast organisms are strains selectedfrom the yeast species Saccharomyces cerevisiae, Saccharomyces kluyveri,Schizosaccharomyces pombe, Sacchoromyces uvarum, Kluyveromyces lactis,Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichiakluyveri, Yarrowia ilpolytica, Candida sp., Candida utilis, Candidacacaoi, Geotrichum sp., and Geotrichum fermentans.

The transformation of the yeast cells may for instance be effected byprotoplast formation followed by transformation in a manner known perse. The medium used to cultivate the cells may be any conventionalmedium suitable for growing yeast organisms. The secreted single-chaininsulin, a significant proportion of which will be present in the mediumin correctly processed form, may be recovered from the medium byconventional procedures including separating the yeast cells from themedium by centrifugation, filtration or catching the insulin precursorby an ion exchange matrix or by a reverse phase absorption matrix,precipitating the proteinaceous components of the supernatant orfiltrate by means of a salt, e.g., ammonium sulphate, followed bypurification by a variety of chromatographic procedures, e.g., ionexchange chromatography, affinity chromatography, or the like.

VI. Methods of Use

In one aspect, the present invention includes a method for maintainingC-peptide levels above the minimum effective therapeutic level in apatient in need thereof, comprising administering to the patient atherapeutic dose of any of the claimed PEGylated C-peptides.

In another aspect, the present invention includes a method for treatingone or more long-term complications of diabetes in a patient in needthereof, comprising administering to the patient a therapeutic dose ofany of the claimed PEGylated C-peptides.

In another aspect, the present invention includes a method for treatinga patient with diabetes comprising administering to the patient atherapeutic dose of any of the claimed PEGylated C-peptides incombination with insulin.

In another aspect, the present invention includes any of the claimedPEGylated C-peptides for use as a C-peptide replacement therapy or dosein a patient in need thereof.

In broad terms, diabetes refers to the situation where the body eitherfails to properly respond to its own insulin, does not make enoughinsulin, or both. The primary result of impaired insulin production isthe accumulation of glucose in the blood, and a C-peptide deficiencyleading to various short- and long-term complications. Three principalforms of diabetes exist:

Type 1: Results from the body's failure to produce insulin andC-peptide. It is estimated that 5-10% of Americans who are diagnosedwith diabetes have type 1 diabetes. Presently almost all persons withtype 1 diabetes must take insulin injections. The term “type 1 diabetes”has replaced several former terms, including childhood-onset diabetes,juvenile diabetes, and insulin-dependent diabetes mellitus (IDDM). Forpatients with type 1 diabetes, basal levels of C-peptide are typicallyless than about 0.20 nM (Ludvigsson et al.: New Engl. J. Med. 359:1909-1920, (2008)).

Type 2: Results from tissue insulin resistance, a condition in whichcells fail to respond properly to insulin, sometimes combined withrelative insulin deficiency. The term “type 2 diabetes” has replacedseveral former terms, including adult-onset diabetes, obesity-relateddiabetes, and non-insulin-dependent diabetes mellitus (NIDDM). For type2 patients in the basal state, C-peptide levels of about 0.8 nM (range0.64 to 1.56 nM), and glucose stimulated levels of about 5.7 nM (range3.7 to 7.7 nM) have been reported. (Retnakaran R et al.: Diabetes Obes.Metab. (2009) DOI 10.11 111/j.1463-1326.2009.01129.x; Zander et al.:Lancet 359: 824-830, (2002)).

In addition to type 1 and type 2 diabetics, there is increasingrecognition of a subclass of diabetes referred to as latent autoimmunediabetes in the adult (LADA) or late-onset autoimmune diabetes ofadulthood, or “slow onset type 1” diabetes, and sometimes also “type1.5” or “type one-and-a-half” diabetes. In this disorder, diabetes onsetgenerally occurs in ages 35 and older, and antibodies against componentsof the insulin-producing cells are always present, demonstrating thatautoimmune activity is an important feature of LADA. It is primarilyantibodies against glutamic acid decarboxylase (GAD) that are found.Some LADA patients show a phenotype similar to that of type 2 patientswith increased body mass index (BMI) or obesity, insulin resistance, andabnormal blood lipids. Genetic features of LADA are similar to those forboth type 1 and type 2 diabetes. During the first 6-12 months afterdebut the patients may not require insulin administration and they areable to maintain relative normoglycemia via dietary modification and/ororal anti-diabetic medication. However, eventually all patients becomeinsulin dependent, probably as a consequence of progressive autoimmuneactivity leading to gradual destruction of the pancreatic islet β-cells.At this stage the LADA patients show low or absent levels of endogenousinsulin and C-peptide, and they are prone to develop long-termcomplications of diabetes involving the peripheral nerves, the kidneys,or the eyes similar to type 1 diabetes patients and thus becomecandidates for C-peptide therapy (Palmer et al.: Diabetes 54(suppl 2):S62-67, (2005); Desai et al.: Diabetic Medicine 25(suppl 2): 30-34,(2008); Fourlanos et al.: Diabetologia 48: 2206-2212, (2005)).

Gestational diabetes: Pregnant women who have never had diabetes beforebut who have high blood sugar (glucose) levels during pregnancy are saidto have gestational diabetes. Gestational diabetes affects about 4% ofall pregnant women. It may precede development of type 2 (or rarelytype 1) diabetes.

Several other forms of diabetes mellitus are categorized separately fromthese. Examples include congenital diabetes due to genetic defects ofinsulin secretion, cystic fibrosis-related diabetes, steroid diabetesinduced by high doses of glucocorticoids, and several forms of monogenicdiabetes.

Accordingly in any of these methods, the term “patient” refers to anindividual who has one of more of the symptoms of any of diabetes. Inone aspect of any of these methods, the term “patient” refers to anindividual who has one of more of the symptoms of any ofinsulin-dependent diabetes. In one aspect of any of these methods, theterm “patient” refers to an individual who has one of more of thesymptoms of any of type 2 diabetes. In one aspect of any of thesemethods, the term “patient” refers to an individual who has one of moreof the symptoms of LADA. In one aspect of any of these methods, the term“patient” refers to an individual who has one of more of the symptoms ofgestational diabetes. Accordingly in one aspect of any of these methods,the term “patient” refers to an individual who has a fasting C-peptidelevel of less than about 0.4 nM. In another aspect of any of thesemethods, the term “patient” refers to an individual who has a fastingC-peptide level of less than about 0.2 nM.

Acute complications of diabetes include hypoglycemia, diabeticketoacidosis, or nonketotic hyperosmolar coma that may occur if thedisease is not adequately controlled. Serious long-term complicationscan also occur, and are discussed in more detail below.

In another aspect, the present invention includes a method for treatingone or more long-term complications of diabetes in a patient in needthereof, comprising administering to the patient a therapeutic dose ofany of the claimed PEGylated C-peptides.

In another aspect, the present invention includes a method for treatinga patient with diabetes comprising administering to the patient atherapeutic dose of any of the claimed PEGylated C-peptides incombination with insulin.

In this context “in combination” means: 1) part of the same unitarydosage form; 2) administration separately, but as part of the sametherapeutic treatment program or regimen, typically but not necessarily,on the same day. In one aspect, any of the claimed PEGylated C-peptidesmay be administered at a fixed daily dosage, and the insulin taken on anas needed basis.

In another aspect, the present invention includes any of the claimedPEGylated C-peptides for use for treating one or more long-termcomplications of diabetes in a patient in need thereof.

In any of these methods, the terms “long-term complication of type 1diabetes”, or “long-term complications of diabetes” refers to thelong-term complications of impaired glycemic control, and C-peptidedeficiency associated with insulin-dependent diabetes. Typicallylong-term complications of type 1 diabetes are associated with type 1diabetics. However the term can also refer to long-term complications ofdiabetes that arise in type 1.5 and type 2 diabetic patients who developa C-peptide deficiency as a consequence of losing pancreatic isletβ-cells and therefore also become insulin dependent. In broad terms,many such complications arise from the primary damage of blood vessels(angiopathy), resulting in subsequent problems that can be grouped under“microvascular disease” (due to damage to small blood vessels) and“macrovascular disease” (due to damage to the arteries).

Specific diseases and disorders included within the term long-termcomplications of diabetes include, without limitation; retinopathyincluding early stage retinopathy with microaneurysms, proliferativeretinopathy, and macular edema; peripheral neuropathy includingsensorimotor polyneuropathy, painful sensory neuropathy, acute motorneuropathy, cranial focal and multifocal polyneuropathies, thoracolumbarradiculoneuropathies, proximal diabetic neuropathies, and focal limbneuropathies including entrapment and compression neuropathies;autonomic neuropathy involving the cardiovascular system, thegastrointestinal tract, the respiratory system, the urigenital system,sudomotor function and papillary function; and nephropathy includingdisorders with microalbuminuria, overt proteinuria, and end-stage renaldisease.

Impaired microcirculatory perfusion appears to be crucial to thepathogenesis of both neuropathy and retinopathy in diabetics. This inturn reflects a hyperglycemia-mediated perturbation of vascularendothelial function that results in: over-activation of protein kinaseC, reduced availability of nitric oxide (NO), increased production ofsuperoxide and endothelin-1 (ET-1), impaired insulin function,diminished synthesis of prostacyclin/PGE1, and increased activation andendothelial adherence of leukocytes. This is ultimately a catastrophicgroup of clinical events.

Accordingly in some embodiments, the term “patient” refers to anindividual who has one of more of the symptoms of the long-termcomplications of diabetes.

Diabetic retinopathy is an ocular manifestation of the systemic damageto small blood vessels leading to microangiopathy. In retinopathy,growth of friable and poor-quality new blood vessels in the retina aswell as macular edema (swelling of the macula) can lead to severe visionloss or blindness. As new blood vessels form at the back of the eye as apart of proliferative diabetic retinopathy (PDR), they can bleed(hemorrhage) and blur vision. It affects up to 80% of all patients whohave had diabetes for 10 years or more.

The symptoms of diabetic retinopathy are often slow to develop andsubtle and include blurred version and progressive loss of sight.Macular edema, which may cause vision loss more rapidly, may not haveany warning signs for some time. In general, however, a person withmacular edema is likely to have blurred vision, making it hard to dothings like read or drive. In some cases, the vision will get better orworse during the day.

Accordingly in some embodiments, the term “patient” refers to anindividual who has one of more of the symptoms of diabetic retinopathy.

Diabetic neuropathies are neuropathic disorders that are associated withdiabetic microvascular injury involving small blood vessels that supplynerves (vasa nervorum). Relatively common conditions which may beassociated with diabetic neuropathy include third nerve palsy;mononeuropathy; mononeuropathy multiplex; diabetic amyotrophy; a painfulpolyneuropathy; autonomic neuropathy; and thoracoabdominal neuropathy.

Diabetic neuropathy affects all peripheral nerves: pain fibers, motorneurons, autonomic nerves. It therefore necessarily can affect allorgans and systems since all are innervated. There are several distinctsyndromes based on the organ systems and members affected, but these areby no means exclusive. A patient can have sensorimotor and autonomicneuropathy or any other combination. Symptoms vary depending on thenerve(s) affected and may include symptoms other than those listed.Symptoms usually develop gradually over years.

Symptoms of diabetic neuropathy may include: numbness and tingling ofextremities, dysesthesia (decreased or loss of sensation to a bodypart), diarrhea, erectile dysfunction, urinary incontinence (loss ofbladder control), impotence, facial, mouth and eyelid drooping, visionchanges, dizziness, muscle weakness, difficulty swallowing, speechimpairment, fasciculation (muscle contractions), anorgasmia, and burningor electric pain.

Additionally, different nerves are affected in different ways byneuropathy. Sensorimotor polyneuropathy, in which longer nerve fibersare affected to a greater degree than shorter ones, because nerveconduction velocity is slowed in proportion to a nerve's length. In thissyndrome, decreased sensation and loss of reflexes occurs first in thetoes on each foot, then extends upward. It is usually described asglove-stocking distribution of numbness, sensory loss, dysesthesia, andnight-time pain. The pain can feel like burning, pricking sensation,achy, or dull. Pins and needles sensation is common. Loss ofproprioception, the sense of where a limb is in space, is affectedearly. These patients cannot feel when they are stepping on a foreignbody, like a splinter, or when they are developing a callous from anill-fitting shoe. Consequently, they are at risk for developing ulcersand infections on the feet and legs, which can lead to amputation.Similarly, these patients can get multiple fractures of the knee, ankle,or foot, and develop a Charcot joint. Loss of motor function results indorsiflexion, contractures of the toes, loss of the interosseous musclefunction, and leads to contraction of the digits, so called hammer toes.These contractures occur not only in the foot, but also in the handwhere the loss of the musculature makes the hand appear gaunt andskeletal. The loss of muscular function is progressive.

Autonomic neuropathy impacts the autonomic nervous system serving theheart, gastrointestinal system, and genitourinary system. The mostcommonly recognized autonomic dysfunction in diabetics is orthostatichypotension, or fainting when standing up. In the case of diabeticautonomic neuropathy, it is due to the failure of the heart and arteriesto appropriately adjust heart rate and vascular tone to keep bloodcontinually and fully flowing to the brain. This symptom is usuallyaccompanied by a loss of the usual change in heart rate seen with normalbreathing. These two findings suggest autonomic neuropathy.

Gastrointestinal system symptoms include delayed gastric emptying,gastroparesis, nausea, bloating, and diarrhea. Because many diabeticstake oral medication for their diabetes, absorption of these medicinesis greatly affected by the delayed gastric emptying. This can lead tohypoglycemia when an oral diabetic agent is taken before a meal and doesnot get absorbed until hours, or sometimes days later, when there isnormal or low blood sugar already. Sluggish movement of the smallintestine can cause bacterial overgrowth, made worse by the presence ofhyperglycemia. This leads to bloating, gas, and diarrhea.

Genitourinary system symptoms include urinary frequency, urgency,incontinence, and retention. Urinary retention can lead to bladderdiverticula, stones, reflux nephropathy, and frequent urinary tractinfections. Accordingly in any of these methods, the term “patient”refers to an individual who has one of more of the symptoms of autonomicneuropathy.

Accordingly in some embodiments, the term “patient” refers to anindividual who has one of more of the symptoms of diabetic neuropathy.In another aspect of any of these methods, the patient has “establishedperipheral neuropathy” which is characterized by reduced sensory nerveconduction velocity (SCV) in the sural nerves (less than −1.5 SD from abody height-corrected reference value for a matched normal individual).In certain embodiments, the term “patient” refers to an individual whohas one of more of the symptoms of incipient neuropathy.

Accordingly in certain embodiments, the current invention includes amethod of treating or preventing a decrease in a subject's, orpatient's, height-adjusted sensory or motor nerve conduction velocity.In one aspect of this method, the motor nerve conduction velocity isinitial nerve conduction velocity. In another embodiment, the motornerve conduction velocity is the peak nerve conduction velocity.

In certain embodiments the subject is a patient with diabetes. Incertain embodiments, the subject has at least one long term complicationof diabetes. In one aspect, the patient exhibits a peak nerve conductionvelocity that is at least about 2 standard deviations from the mean peaknerve conduction velocity for a similar height-matched subject group. Inone aspect, the patients have a peak nerve conduction velocity ofgreater than about 35 m/s. In one aspect of any of the claimed methods,the patients have a peak nerve conduction velocity of greater than about40 m/s. In one aspect, the patients have a peak nerve conductionvelocity of greater than about 45 m/s. In one aspect, the patients havea peak nerve conduction velocity of greater than about 50 m/s.

In one aspect of any of the claimed methods, treatment results in animprovement in nerve conduction velocity of at least about 1.5 m/s. Inanother aspect of these methods, treatment results in an improvement innerve conduction velocity of at least about 2.0 m/s. In another aspectof these methods, treatment results in an improvement in nerveconduction velocity of at least about 2.5 m/s. In another aspect ofthese methods, treatment results in an improvement in nerve conductionvelocity of at least about 3.0 m/s. In another aspect of these methods,treatment results in an improvement in nerve conduction velocity of atleast about 3.5 m/s. In another aspect of these methods, treatmentresults in an improvement in nerve conduction velocity of at least about4.0 m/s. In another aspect of these methods, treatment results in animprovement in nerve conduction velocity of at least about 4.5 m/s. Inanother aspect of these methods, treatment results in an improvement innerve conduction velocity of at least about 5.0 m/s. In another aspectof these methods, treatment results in an improvement in nerveconduction velocity of at least about 5.5 m/s. In another aspect ofthese methods, treatment results in an improvement in nerve conductionvelocity of at least about 6.0 m/s. In another aspect of these methods,treatment results in an improvement in nerve conduction velocity of atleast about 7.0 m/s. In another aspect of these methods, treatmentresults in an improvement in nerve conduction velocity of at least about8.0 m/s. In another aspect of these methods, treatment results in animprovement in nerve conduction velocity of at least about 9.0 m/s. Inanother aspect of these methods, treatment results in an improvement innerve conduction velocity of at least about 10.0 m/s. In another aspectof these methods, treatment results in an improvement in nerveconduction velocity of at least about 15.0 m/s. In another aspect ofthese methods, treatment results in an improvement in nerve conductionvelocity of at least about 20.0 m/s.

In certain embodiments, of any of these methods, treatment results in animprovement of at least 10% in peak nerve conduction velocity comparedto peak nerve conduction velocity prior to starting PEGylated C-peptidetherapy. In certain embodiments, of any of these methods, treatmentresults in an improvement of at least 15% in peak nerve conductionvelocity compared to peak nerve conduction velocity prior to startingPEGylated C-peptide therapy. In certain embodiments, of any of thesemethods, treatment results in an improvement of at least 20% in peaknerve conduction velocity compared to peak nerve conduction velocityprior to starting PEGylated C-peptide therapy. In certain embodiments,of any of these methods, treatment results in an improvement of at least25% in peak nerve conduction velocity compared to peak nerve conductionvelocity prior to starting PEGylated C-peptide therapy. In certainembodiments, of any of these methods, treatment results in animprovement of at least 30% in peak nerve conduction velocity comparedto peak nerve conduction velocity prior to starting PEGylated C-peptidetherapy. In certain embodiments, of any of these methods, treatmentresults in an improvement of at least 40% in peak nerve conductionvelocity compared to peak nerve conduction velocity prior to startingPEGylated C-peptide therapy. In certain embodiments, of any of thesemethods, treatment results in an improvement of at least 50% in peaknerve conduction velocity compared to peak nerve conduction velocityprior to starting PEGylated C-peptide therapy.

Diabetic nephropathy is a progressive kidney disease caused byangiopathy of capillaries in the kidney glomeruli. It is characterizedby nephrotic syndrome and diffuse glomerulosclerosis. It is due tolong-standing diabetes mellitus, and is a prime cause for dialysis inmany Western countries.

The symptoms of diabetic nephropathy can be seen in patients withchronic diabetes (15 years or more after onset). The disease isprogressive and is more frequent in men. Diabetic nephropathy is themost common cause of chronic kidney failure and end-stage kidney diseasein the United States. People with both type 1 and type 2 diabetes are atrisk. The risk is higher if blood-glucose levels are poorly controlled.Further, once nephropathy develops, the greatest rate of progression isseen in patients with poor control of their blood pressure. Also peoplewith high cholesterol level in their blood have much more risk thanothers.

The earliest detectable change in the course of diabetic nephropathy isan abnormality of the glomerular filtration barrier. At this stage, thekidney may start allowing more serum albumin than normal in the urine(albuminuria), and this can be detected by sensitive medical tests foralbumin. This stage is called “microalbuminuria”. As diabeticnephropathy progresses, increasing numbers of glomeruli are destroyed bynodular glomerulosclerosis. Now the amounts of albumin being excreted inthe urine increases, and may be detected by ordinary urinalysistechniques. At this stage, a kidney biopsy clearly shows diabeticnephropathy.

Kidney failure provoked by glomerulosclerosis leads to fluid filtrationdeficits and other disorders of kidney function. There is an increase inblood pressure (hypertension) and fluid retention in the body plus areduced plasma oncotic pressure causes edema. Other complications may bearteriosclerosis of the renal artery and proteinuria.

Throughout its early course, diabetic nephropathy has no symptoms. Theydevelop in late stages and may be a result of excretion of high amountsof protein in the urine or due to renal failure. Symptoms include,edema; swelling, usually around the eyes in the mornings; later, generalbody swelling may result, such as swelling of the legs, foamy appearanceor excessive frothing of the urine (caused by the proteinura),unintentional weight gain (from fluid accumulation), anorexia (poorappetite), nausea and vomiting, malaise (general ill feeling), fatigue,headache, frequent hiccups, and generalized itching.

Accordingly in some embodiments, the term “patient” refers to anindividual who has one of more of the symptoms of diabetic nephropathy.

Diabetic cardiomyopathy (DCM), damage to the heart, leading to diastolicdysfunction and eventually heart failure. Aside from large vesseldisease and accelerated atherosclerosis, which is very common indiabetes, DCM is a clinical condition diagnosed when ventriculardysfunction develops in patients with diabetes in the absence ofcoronary atherosclerosis and hypertension. DCM may be characterizedfunctionally by ventricular dilation, myocyte hypertrophy, prominentinterstitial fibrosis, and decreased or preserved systolic function inthe presence of a diastolic dysfunction.

One particularity of DCM is the long latent phase, during which thedisease progresses but is completely asymptomatic. In most cases, DCM isdetected with concomitant hypertension or coronary artery disease. Oneof the earliest signs is mild left ventricular diastolic dysfunctionwith little effect on ventricular filling. Also, the diabetic patientmay show subtle signs of DCM related to decreased left ventricularcompliance or left ventricular hypertrophy or a combination of both. Aprominent “a” wave can also be noted in the jugular venous pulse, andthe cardiac apical impulse may be overactive or sustained throughoutsystole. After the development of systolic dysfunction, left ventriculardilation and symptomatic heart failure, the jugular venous pressure maybecome elevated and the apical impulse would be displaced downward andto the left. Systolic mitral murmur is not uncommon in these cases.These changes are accompanied by a variety of electrocardiographicchanges that may be associated with DCM in 60% of patients withoutstructural heart disease, although usually not in the early asymptomaticphase. Later in the progression, a prolonged QT interval may beindicative of fibrosis. Given that the definition of DCM excludesconcomitant atherosclerosis or hypertension, there are no changes inperfusion or in atrial natriuretic peptide levels up until the very latestages of the disease, when the hypertrophy and fibrosis become verypronounced.

In certain embodiments, the term “patient” refers to an individual whohas one of more of the symptoms of diabetic cardiomyopathy.

Macrovascular diseases of diabetes include coronary artery disease,leading to angina or myocardial infarction (“heart attack”), stroke(mainly the ischemic type), peripheral vascular disease, whichcontributes to intermittent claudication (exertion-related leg and footpain), as well as diabetic foot and diabetic myonecrosis (“musclewasting”).

In certain embodiments, the term “patient” refers to an individual whohas one of more of the symptoms of a macrovascular disease of diabetes.

Methods for Preventing Hypoglycemia.

In certain embodiments, the present invention includes the use of any ofthe disclosed PEGylated C-peptides to reduce the risk of hypoglycemia ina human patient with insulin dependent diabetes, in a regimen whichadditionally comprises the administration of insulin, comprising; a)administering insulin to said patient; b) administering a therapeuticdose of PEGylated C-peptide in a different site as that used for saidpatient's insulin administration; c) adjusting the dosage amount, type,or frequency of insulin administered based on said patient's alteredinsulin requirements resulting from said therapeutic dose of PEGylatedC-peptide.

In another aspect, the present invention includes a method of reducinginsulin usage in an insulin-dependent human patient, comprising thesteps of; a) administering insulin to said patient; b) administeringsubcutaneously to said patient a therapeutic dose of any of thedisclosed PEGylated C-peptides in a different site as that used for saidpatient's insulin administration; c) adjusting the dosage amount, type,or frequency of insulin administered based on monitoring said patient'saltered insulin requirements resulting from said therapeutic dose ofPEGylated C-peptide, wherein said adjusted dose of insulin does notinduce hyperglycemia, wherein said adjusted dose of insulin is at least10% less than said patient's insulin dose prior to starting PEGylatedC-peptide. (See for example U.S. Pat. No. 7,855,177, which is hereinincorporated by reference).

In any of these methods, the term “hypoglycemia” or “hypoglycemicevents” refers to all episodes of abnormally low plasma glucoseconcentration that exposes the patient to potential harm. The AmericanDiabetes Association Workgroup has recommended that people withinsulin-dependent diabetes become concerned about the possibility ofdeveloping hypoglycemia at a plasma glucose concentration of less than70 mg/dL (3.9 mmoL/L). Accordingly in one aspect of any of the claimedmethods, the terms hypoglycemia or hypoglycemic event refers to thesituation where the plasma glucose concentration of the patient drops toless than about 70 mg/dL (3.9 mmoL/L).

Hypoglycemia is a serious medical complication in the treatment ofdiabetes, and causes recurrent morbidity in most people with type 1diabetes and many with advanced type 2 diabetes and is sometimes fatal.In addition, hypoglycemia compromises physiological and behavioraldefenses against subsequent falling plasma glucose concentrations andthus causes a vicious cycle of recurrent hypoglycemia. Accordingly theprevention of hypoglycemia is of significant importance in the treatmentof diabetes, as well as the treatment of the long-term complications ofdiabetes.

Unfortunately hypoglycemia is a fact of life for most people with type 1diabetes (Cryer P E et al.: Diabetes 57: 3169-3176, (2008)). The averagepatient has untold numbers of episodes of asymptomatic hypoglycemia andsuffers two episodes of symptomatic hypoglycemia per week, withthousands of such episodes over a lifetime of diabetes. He or shesuffers one or more episodes of severe, temporarily disablinghypoglycemia often with seizure or coma, per year.

Overall, hypoglycemia is less frequent in type 2 diabetes; however, therisk of hypoglycemia becomes progressively more frequent and limiting toglycemic control later in the course of type 2 diabetes. Theprospective, population-based data of Donnelly et al. (Diabetes Med. 22:749-755, (2005)) indicate that the overall incidence of hypoglycemia ininsulin-treated type 2 diabetes is approximately one third of that intype 1 diabetes. The incidence of any hypoglycemia and of severehypoglycemia was 4,300 and 115 episodes per 100 patient years,respectively, in type 1 diabetes and 1600 and 35 episodes per 100patient years, respectively, in insulin-treated type 2 diabetes.

Hypoglycemia may be classified based on the severity of the hypoglycemicevent. For example, the American Diabetes Association Workgroup hassuggested the following classification of hypoglycemia in diabetes: 1)severe hypoglycemia (i.e., hypoglycemic coma requiring assistance ofanother person); 2) documented symptomatic hypoglycemia (with symptomsand a plasma glucose concentration of less than 70 mg/dL); 3)asymptomatic hypoglycemia (with a plasma glucose concentration of lessthan 70 mg/dL without symptoms); 4) probable symptomatic hypoglycemia(with symptoms attributed to hypoglycemia, but without a plasma glucosemeasurement); and 5) relative hypoglycemia (with a plasma glucoseconcentration of greater than 70 mg/dL but falling towards that level).

Thus in another aspect of any of the methods disclosed herein, the term“hypoglycemia” refers to severe hypoglycemia, and/or hypoglycemic coma.In another aspect of any of these methods, the term “hypoglycemia”refers to symptomatic hypoglycemia. In another aspect of any of thesemethods, the term “hypoglycemia” refers to probable symptomatichypoglycemia. In another aspect of any of these methods, the term“hypoglycemia” refers to asymptomatic hypoglycemia. In another aspect ofany of these methods, the term “hypoglycemia” refers to relativehypoglycemia.

Insulin Types and Administration Forms

There are over 180 individual insulin preparations available worldwidewhich have been developed to provide different lengths of activity(activity profiles). Approximately 25% of these are soluble insulin(unmodified form); about 35% are long- or intermediate-acting basalinsulins (mixed with NPH [neutral protamine Hagedorn] insulin or Lenteinsulin [insulin zinc suspension], or forms that are modified to have anincreased isoelectric point [insulin glargine], or acylation [insulindetemir]; these forms have reduced solubility, slow subcutaneousabsorption, and long duration of action relative to soluble insulins);about 2% are rapid-acting insulins (e.g., which are engineered by aminoacid change, and have reduced self-association and increasedsubcutaneous absorption); and about 38% are pre-mixed insulins (e.g.,mixtures of short-, intermediate-, and long-acting insulins; thesepreparations have the benefit of a reduced number of daily injections).

Short-acting insulin preparations that are commercially available in theUS include regular insulin and rapid-acting insulins. Regular insulinhas an onset of action of 30-60 minutes, peak time of effect of 1.5 to 2hours, and duration of activity of 5 to 12 hours. Rapid-acting insulins,such as Aspart (Novo Rapid), Lispro (Humalog), and Glulisine (Apidra),have an onset of action of 10-30 minutes, peak time of effect of around30 minutes, and a duration of activity of 3 to 5 hours.

Intermediate-acting insulins, such as NPH and Lente insulins, have anonset of action of 1 to 2 hours, peak time of effect of 4 to 8 hours,and a duration of activity of 10 to 20 hours.

Long-acting insulins, such as Ultralente insulin, have an onset ofaction of 2 to 4 hours, peak time of effect of 8 to 20 hours, and aduration of activity of 16 to 24 hours. Other examples of long-actinginsulins include Glargine and Determir. Glargine insulin has an onset ofaction of 1 to 2 hours, and a duration of action of 24 hours, but withno peak effect.

In many cases, regimens that use insulin in the management of diabetescombine long-acting and short-acting insulin. For example, Lantus, fromAventis Pharmaceuticals Inc., is a recombinant human insulin analog thatis a long-acting, parenteral blood-glucose-lowering agent whose longerduration of action (up to 24 hours) is directly related to its slowerrate of absorption. Lantus is administered subcutaneously once a day,preferably at bedtime, and is said to provide a continuous level ofinsulin, similar to the slow, steady (basal) secretion of insulinprovided by the normal pancreas. The activity of such a long-actinginsulin results in a relatively constant concentration/time profile over24 hours with no pronounced peak, thus allowing it to be administeredonce a day as a patient's basal insulin. Such long-acting insulin has along-acting effect by virtue of its chemical composition, rather than byvirtue of an addition to insulin when administered.

More recently automated wireless controlled systems for continuousinfusion of insulin, such as the system sold under the trademarkOMNIPOD™ Insulin Management System (Insulet Corporation, Bedford, Mass.)have been developed. These systems provide continuous subcutaneousinsulin delivery with blood glucose monitoring technology in a discreettwo-part system. This system eliminates the need for daily insulininjections, and does not require a conventional insulin pump which isconnected via tubing.

OMNIPOD™ is a small lightweight device that is worn on the skin like aninfusion set. It delivers insulin according to pre-programmedinstructions transmitted wirelessly from the Personal Diabetes Manager(PDM). The PDM is a wireless, hand-held device that is used to programthe OMNIPOD™ Insulin Management System with customized insulin deliveryinstructions, monitor the operation of the system, and check bloodglucose levels using blood glucose test strips sold under the trademarkFREESTYLE™. There is no tubing connecting the device to the PDM.OMNIPOD™ Insulin Management System is worn beneath the clothing, and thePDM can be carried separately in a backpack, briefcase, or purse.Similar to currently available insulin pumps, the OMNIPOD™ InsulinManagement System features fully programmable continuous subcutaneousinsulin delivery with multiple basal rates and bolus options, suggestedbolus calculations, safety checks, and alarm features.

The aim of insulin treatment of diabetics is typically to administerenough insulin such that the patient will have blood glucose levelswithin the physiological range and normal carbohydrate metabolismthroughout the day. Because the pancreas of a diabetic individual doesnot secrete sufficient insulin throughout the day, in order toeffectively control diabetes through insulin therapy, a long-lastinginsulin treatment, known as basal insulin, must be administered toprovide the slow and steady release of insulin that is needed to controlblood glucose concentrations and to keep cells supplied with energy whenno food is being digested. Basal insulin is necessary to suppressglucose production between meals and overnight and preferably mimics thepatient's normal pancreatic basal insulin secretion over a 24-hourperiod. Thus, a diabetic patient may administer a single dose of along-acting insulin each day subcutaneously, with an action lastingabout 24 hours.

Furthermore, in order to effectively control diabetes through insulintherapy by dealing with postprandial rises in glucose levels, a bolus,fast-acting treatment must also be administered. The bolus insulin,which is generally administered subcutaneously, provides a rise inplasma insulin levels at approximately 1 hour after administration,thereby limiting hyperglycemia after meals. Thus, these additionalquantities of regular insulin, with a duration of action of, e.g., 5 to6 hours, may be subcutaneously administered at those times of the daywhen the patient's blood glucose level tends to rise too high, such asat meal times. As an alternative to administering basal insulin incombination with bolus insulin, repeated and regular lower doses ofbolus insulin may be administered in place of the long-acting basalinsulin, and bolus insulin may be administered postprandially as needed.

Currently, regular subcutaneously injected insulin is recommended to bedosed at 30 to 45 minutes prior to mealtime. As a result, diabeticpatients and other insulin users must engage in considerable planning oftheir meals and of their insulin administrations relative to theirmeals. Unfortunately, intervening events that may take place betweenadministration of insulin and ingestion of the meal may affect theanticipated glucose excursions.

Furthermore, there is also the potential for hypoglycemia if theadministered insulin provides a therapeutic effect over too great atime, e.g., after the rise in glucose levels that occur as a result ofingestion of the meal has already been lowered. As outlined in theExamples, this risk of hypoglycemia is increased in patients who havebeen treated with C-peptide due to a reduced requirement for insulin.

Accordingly, in one aspect of any of the methods disclosed herein, thepresent invention includes a method for reducing the risk of the patientdeveloping hypoglycemia by reducing the average daily dose of insulinadministered to the patient by about 5% to about 50% after startingPEGylated C-peptide therapy. In another aspect, the dose of insulinadministered is reduced by about 5% to about 45% compared to thepatient's insulin dose prior to starting PEGylated C-peptide treatment.In another aspect, the dose of insulin administered is reduced by about5% to about 40% compared to the patient's insulin dose prior to startingPEGylated C-peptide treatment. In another aspect, the dose of insulinadministered is reduced by about 5% to about 35% compared to thepatient's insulin dose prior to starting PEGylated C-peptide treatment.In another aspect, the dose of insulin administered is reduced by about5% to about 30% compared to the patient's insulin dose prior to startingPEGylated C-peptide treatment. In another aspect, the dose of insulinadministered is reduced by about 5% to about 25% compared to thepatient's insulin dose prior to starting PEGylated C-peptide treatment.In another aspect, the dose of insulin administered is reduced by about5% to about 20% compared to the patient's insulin dose prior to startingPEGylated C-peptide treatment. In another aspect, the dose of insulinadministered is reduced by about 5% to about 15% compared to thepatient's insulin dose prior to starting PEGylated C-peptide treatment.In another aspect, the dose of insulin administered is reduced by about5% to about 10% compared to the patient's insulin dose prior to startingPEGylated C-peptide treatment.

In another aspect, the dose of insulin administered is reduced by about2% to about 10% compared to the patient's insulin dose prior to startingPEGylated C-peptide treatment. In another aspect, the dose of insulinadministered is reduced by about 2% to about 15% compared to thepatient's insulin dose prior to starting PEGylated C-peptide treatment.In another aspect, the dose of insulin administered is reduced by about2% to about 20% compared to the patient's insulin dose prior to startingPEGylated C-peptide treatment.

In another aspect, the dose of insulin administered is reduced by about10% to about 50% compared to the patient's insulin dose prior tostarting PEGylated C-peptide treatment. In another aspect, the dose ofinsulin administered is reduced by about 10% to about 45% compared tothe patient's insulin dose prior to starting PEGylated C-peptidetreatment. In another aspect, the dose of insulin administered isreduced by about 10% to about 40% compared to the patient's insulin doseprior to starting PEGylated C-peptide treatment. In another aspect, thedose of insulin administered is reduced by about 10% to about 35%compared to the patient's insulin dose prior to starting C-peptidetreatment. In another aspect, the dose of insulin administered isreduced by about 10% to about 30% compared to the patient's insulin doseprior to starting PEGylated C-peptide treatment. In another aspect, thedose of insulin administered is reduced by about 10% to about 25%compared to the patient's insulin dose prior to starting PEGylatedC-peptide treatment. In another aspect, the dose of insulin administeredis reduced by about 10% to about 20% compared to the patient's insulindose prior to starting PEGylated C-peptide treatment. In another aspect,the dose of insulin administered is reduced by at least 10% compared tothe patient's insulin dose prior to starting PEGylated C-peptidetreatment.

In one aspect of any of these methods, the dose of short-acting insulinadministered is selectively reduced by any of the prescribed rangeslisted above. In another aspect of any of these methods, the dose ofintermediate-acting insulin administered is selectively reduced by anyof the prescribed ranges. In one aspect of any of these methods, thedose of long-acting insulin administered is selectively reduced by anyof the prescribed ranges listed above.

In another aspect of any of these methods, the dose of intermediate- andlong-acting insulin administered is independently reduced by any of theprescribed ranges listed above, while the dose of short-acting insulinremains substantially unchanged.

In one aspect of these methods, the dose of short-acting insulinadministered is reduced by about 5% to about 50% compared to thepatient's insulin dose prior to starting PEGylated C-peptide treatment.In another embodiment, the dose of short-acting insulin administered isreduced by about 5% to about 35% compared to the patient's insulin doseprior to starting PEGylated C-peptide treatment. In another embodiment,the dose of short-acting insulin administered is reduced by about 10% toabout 20% compared to the patient's insulin dose prior to startingPEGylated C-peptide treatment. In one aspect of these methods, the doseof short-acting insulin administered preprandially for a meal isreduced. In another aspect of these methods, the dose of short-actinginsulin administered in the morning or at nighttime is reduced. Inanother aspect of any of these methods, the dose of short-acting insulinadministered is reduced while the dose of long-acting and/orintermediate-acting insulin administered to the patient is substantiallyunchanged.

In another aspect of any of the methods disclosed herein, the presentinvention includes a method for reducing the risk of the patientdeveloping hypoglycemia by reducing the average daily dose ofintermediate-acting insulin administered to the patient by about 5% toabout 35% after starting PEGylated C-peptide therapy. In one aspect ofthese methods, the dose of intermediate-acting insulin administered isreduced by about 5% to about 50% compared to the patient's insulin doseprior to starting PEGylated C-peptide treatment. In another embodiment,the dose of intermediate-acting insulin administration is reduced byabout 5% to about 35% compared to the patient's insulin dose prior tostarting PEGylated C-peptide treatment. In another embodiment, the doseof intermediate-acting insulin administered is reduced by about 10% toabout 20% compared to the patient's insulin dose prior to startingPEGylated C-peptide treatment. In another aspect of these methods, thedose of intermediate-acting insulin administered in the morning or atnighttime is reduced. In another aspect of any of these methods, thedose of intermediate-acting insulin administered is reduced while thedose of short-acting insulin administered to the patient issubstantially unchanged.

In another aspect of any of the methods disclosed herein, the presentinvention includes a method for reducing the risk of the patientdeveloping hypoglycemia by reducing the average daily dose oflong-acting insulin administered to the patient by about 5% to about 50%after starting PEGylated C-peptide therapy. In one embodiment, the doseof long-acting insulin administered is reduced by about 5% to about 35%compared to the patient's insulin dose prior to starting PEGylatedC-peptide treatment. In another embodiment, the dose of long-actinginsulin administered is reduced by about 10% to about 20% compared tothe patient's insulin dose prior to starting PEGylated C-peptidetreatment. In another aspect of these methods, the dose of long-actinginsulin administered in the morning or at nighttime is reduced. Inanother aspect of any of these methods, the dose of long-acting insulinadministered is reduced while the dose of short-acting insulinadministered to the patient is substantially unchanged.

In certain preferred embodiments, the patient achieves improved insulinutilization and insulin sensitivity while experiencing a reduced risk ofdeveloping hypoglycemia after treatment with PEGylated C-peptide ascompared with baseline levels prior to treatment. Preferably, theimproved insulin utilization and insulin sensitivity are measured by astatistically significant decline in HOMA (Homeostasis Model Assessment)(Turner et al.: Metabolism 28(11): 1086-1096, (1979)).

Subcutaneous administration of the PEGylated C-peptide will typicallynot be into the same site as that most recently used for insulinadministration, i.e. PEGylated C-peptide and insulin will be injectedinto different sites. Specifically in one aspect, the site of PEGylatedadministration will typically be at least about 10 cm way from the sitemost recently used for insulin administration. In another aspect, thesite of PEGylated C-peptide administration will typically be at leastabout 15 cm away from the site most recently used for insulinadministration. In another aspect, the site of PEGylated C-peptideadministration will typically be at least about 20 cm away from the sitemost recently used for insulin administration.

Examples of different sites include for example, and without limitation,injections into the left and right arm, or injections into the left andright thigh, or injections into the left or right buttock, or injectionsinto the opposite sides of the abdomen. Other obvious variants ofdifferent sites include injections in an arm and thigh, or injections inan arm and buttock, or injections into an arm and abdomen, etc.

Moreover one of ordinary skill in the art, i.e. a physician, or diabeticpatient, will recognize and understand how to inject PEGylated C-peptideand insulin into any other combination of different sites, based on theprior art teaching, and numerous text books and guides on insulinadministration that provide disclosure on how to select differentinsulin injection sites. See for example, the following representativetext books (Learning to live well with diabetes, Ed. Cheryl Weiler,(1991) DCI Publishing, Minneapolis, Minn.; American Diabetes AssociationComplete Guide to Diabetes, ISBN 0-945448-64-3 (1996)).

In one aspect of any of the claimed methods, PEGylated C-peptide isadministered to the opposite side of the abdomen to the site mostrecently used for insulin administration, approximately 15 to 20 cmapart.

VII. Pharmaceutical Compositions

In one aspect, the present invention includes a pharmaceuticalcomposition comprising PEGylated C-peptide, and a pharmaceuticallyacceptable carrier, diluent or excipient.

Pharmaceutical compositions suitable for the delivery of PEGylatedC-peptide and methods for their preparation will be readily apparent tothose skilled in the art and may comprise any of the known carriers,diluents, or excipients. Such compositions and methods for theirpreparation may be found, e.g., in Remington's Pharmaceutical Sciences,19th Edition (Mack Publishing Company, 1995).

In one aspect, the pharmaceutical compositions may be in the form ofsterile aqueous solutions and/or suspensions of the pharmaceuticallyactive ingredients, aerosols, ointments, and the like. Formulationswhich are aqueous solutions are most preferred. Such formulationstypically contain the PEGylated C-peptide itself, water, and one or morebuffers which act as stabilizers (e.g., phosphate-containing buffers)and optionally one or more preservatives. Such formulations containing,e.g., about 1 to 200 mg, about 3 to 100 mg, about 3 to 80 mg, about 3 to60 mg, about 3 to 40 mg, about 3 to 30 mg, about 0.3 to 3.3 mg, about 1to 3.3 mg, about 1 to 2 mg, about 1 to 3.3 mg, about 2 to 3.3 mg or anyof the ranges mentioned herein, e.g., about 200 mg, about 150 mg, about120 mg, about 100 mg, about 80 mg, about 60 mg, about 50 mg, about 40mg, about 30 mg, about 20 mg, or about 10 mg, or about 8 mg, or about 6mg, or about 5 mg, or about 4 mg, or about 3 mg, or about 2 mg, or about1 mg, or about 0.5 mg of the PEGylated C-peptide and constitute afurther aspect of the invention.

Pharmaceutical compositions may include pharmaceutically acceptablesalts of PEGylated C-peptide. For a review on suitable salts, seeHandbook of Pharmaceutical Salts: Properties, Selection, and Use byStahl and Wermuth (Wiley-VCH, 2002). Suitable base salts are formed frombases which form non-toxic salts. Representative examples include thealuminium, arginine, benzathine, calcium, choline, diethylamine,diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium,sodium, tromethamine, and zinc salts. Hemisalts of acids and bases mayalso be formed, e.g., hemisulphate and hemicalcium salts. In oneembodiment, PEGylated C-peptide may be prepared as a gel with apharmaceutically acceptable positively charged ion.

In one aspect, the positively charged ion may be a monovalent metal ion.In one aspect, the metal ion is selected from sodium and potassium.

In one aspect, the positively charged ion may be a divalent metal ion.In one aspect, the metal ion is selected from calcium, magnesium, andzinc.

The PEGylated C-peptide may be administered at any time during the day.For humans, the dosage used may range from about 0.1 to 200 mg/week ofPEGylated C-peptide, e.g., from about 0.1 to 0.3 mg/week, about 0.3 to1.5 mg/week, about 1 mg to about 3.5 mg/week, about 1.5 to 2.25 mg/week,about 2.25 to 3.0 mg/week, about 3.0 to 6.0 mg/week, about 6.0 to 10mg/week, about 10 to 20 mg/week, about 20 to 40 mg/week, about 40 to 60mg/week, about 60 to 80 mg/week, about 80 to 100 mg/week, about 100 to120 mg/week, about 120 to 140 mg/week, about 140 to 160 mg/week, about160 to 180 mg/week, and about 180 to about 200 mg/week.

Preferably the total weekly dose used of PEGylated C-peptide is about 1mg to about 3.5 mg, about 1 mg to about 20 mg, about 20 mg to about 50mg, about 50 mg to about 100 mg, about 100 mg to about 150 mg, or about150 mg to about 200 mg.

The total weekly dose of PEGylated C-peptide may be about 0.1 mg, about0.5 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg,about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 12 mg,about 15 mg, about 18 mg, about 21 mg, about 24 mg, about 27 mg, about30 mg, about 33 mg, about 36 mg, about 39 mg, about 42 mg, about 45 mg,about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, or about200 mg. (It will be appreciated that masses of PEGylated C-peptidereferred to above are dependent on the bioavailability of the deliverysystem and based on the use of PEGylated C-peptide with a molecular massof approximately 40,000 Da.)

In another aspect of any of these methods and pharmaceuticalcompositions, the therapeutic dose of PEGylated C-peptide comprises aweekly dose ranging from about 1 mg to about 45 mg. In another aspect ofany of these methods and pharmaceutical compositions, the therapeuticdose of PEGylated C-peptide comprises a weekly dose ranging from about 3mg to about 15 mg. In another aspect of any of these methods andpharmaceutical compositions, the therapeutic dose of PEGylated C-peptidecomprises a weekly dose ranging from about 30 mg to about 60 mg. Inanother aspect of any of these methods and pharmaceutical compositions,the therapeutic dose of PEGylated C-peptide comprises a weekly doseranging from about 60 mg to about 120 mg.

In another aspect of any of these methods and pharmaceuticalcompositions, the therapeutic dose of PEGylated C-peptide maintains theaverage steady-state concentration of PEGylated C-peptide (C_(ss-ave))in the patient's plasma of between about 0.2 nM and about 6 nM.

In another aspect of any of these methods and pharmaceuticalcompositions, the therapeutic dose of PEGylated C-peptide is provided tothe patient so as to maintain the average steady-state concentration ofPEGylated C-peptide in the patient's plasma between about 0.2 nM andabout 6 nM when using a dosing interval of 3 days or longer. In anotheraspect of any of these methods and pharmaceutical compositions, thetherapeutic dose of PEGylated C-peptide is provided to the patient so asto maintain the average steady-state concentration of PEGylatedC-peptide in the patient's plasma between about 0.2 nM and about 6 nMwhen using a dosing interval of 4 days or longer. In another aspect ofany of these methods and pharmaceutical compositions, the therapeuticdose of PEGylated C-peptide is provided to the patient so as to maintainthe average steady-state concentration of PEGylated C-peptide in thepatient's plasma between about 0.2 nM and about 6 nM when using a dosinginterval of 5 days or longer. In another aspect of any of these methodsand pharmaceutical compositions, the therapeutic dose of PEGylatedC-peptide is provided to the patient so as to maintain the averagesteady-state concentration of PEGylated C-peptide in the patient'splasma between about 0.2 nM and about 6 nM when using a dosing intervalof at least one week. In any of these methods and pharmaceuticalcompositions, the therapeutic dose is administered by daily subcutaneousinjections. In another aspect of any of these methods and pharmaceuticalcompositions, the therapeutic dose is administered by a sustainedrelease formulation or device.

In another aspect of any of these methods and pharmaceuticalcompositions, the therapeutic dose of PEGylated C-peptide is provided tothe patient so as to maintain the average steady-state concentration ofPEGylated C-peptide in the patient's plasma between about 0.4 nM andabout 8 nM when using a dosing interval of 3 days or longer. In anotheraspect of any of these methods and pharmaceutical compositions, thetherapeutic dose of PEGylated C-peptide is provided to the patient so asto maintain the average steady-state concentration of PEGylatedC-peptide in the patient's plasma between about 0.4 nM and about 8 nMwhen using a dosing interval of 4 days or longer. In another aspect ofany of these methods and pharmaceutical compositions, the therapeuticdose of PEGylated C-peptide is provided to the patient so as to maintainthe average steady-state concentration of PEGylated C-peptide in thepatient's plasma between about 0.4 nM and about 8 nM when using a dosinginterval of 5 days or longer. In another aspect of any of these methodsand pharmaceutical compositions, the therapeutic dose of PEGylatedC-peptide is provided to the patient so as to maintain the averagesteady-state concentration of PEGylated C-peptide in the patient'splasma between about 0.4 nM and about 8 nM when using a dosing intervalof 7 days or longer.

In another aspect of any of these methods and pharmaceuticalcompositions, the therapeutic dose of PEGylated C-peptide is provided tothe patient so as to maintain the average steady-state concentration ofPEGylated C-peptide in the patient's plasma between about 0.6 nM andabout 8 nM when using a dosing interval of 3 days or longer. In anotheraspect of any of these methods and pharmaceutical compositions, thetherapeutic dose of PEGylated C-peptide is provided to the patient so asto maintain the average steady-state concentration of PEGylatedC-peptide in the patient's plasma between about 0.6 nM and about 8 nMwhen using a dosing interval of 4 days or longer. In another aspect ofany of these methods and pharmaceutical compositions, the therapeuticdose of PEGylated C-peptide is provided to the patient so as to maintainthe average steady-state concentration of PEGylated C-peptide in thepatient's plasma between about 0.6 nM and about 8 nM when using a dosinginterval of 5 days or longer. In another aspect of any of these methodsand pharmaceutical compositions, the therapeutic dose of PEGylatedC-peptide is provided to the patient so as to maintain the averagesteady-state concentration of PEGylated C-peptide in the patient'splasma between about 0.6 nM and about 8 nM when using a dosing intervalof 7 days or longer.

The dose may or may not be in solution. If the dose is administered insolution, it will be appreciated that the volume of the dose may vary,but will typically be 20 μL-2 mL. Preferably the dose for S.C.administration will be given in a volume of 2000 μL, 1500 μL, 1200 μL,1000 μL, 900 μL, 800 μL, 700 μL, 600 μL, 500 μL, 400 μL, 300 μL, 200 μL,100 μL, 50 μL, or 20 μL.

PEGylated C-peptide doses in solution can also comprise a preservativeand/or a buffer. For example, the preservatives m-cresol, or phenol canbe used. Typical concentrations of preservatives include 0.5 mg/mL, 1mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, or 5 mg/mL. Thus, a range ofconcentration of preservative may include 0.2 to 10 mg/mL, particularly0.5 to 6 mg/mL, or 0.5 to 5 mg/mL. Examples of buffers that can be usedinclude histidine (pH 6.0), sodium phosphate buffer (pH 6 to 7.5), orsodium bicarbonate buffer (pH 7 to 7.5). It will be appreciated that thePEGylated C-peptide dose may comprise one or more of a native or intactC-peptide, fragments, derivatives, or other functionally equivalentvariants of C-peptide.

VIII. Methods for Administration

A dose of PEGylated C-peptide may comprise full-length human C-peptide(SEQ. ID. No. 1) and the C-terminal C-peptide fragment EGSLQ (SEQ. ID.No. 31) and/or a C-peptide homolog or C-peptide derivative. Further, thedose may if desired only contain a fragment of C-peptide, e.g., EGSLQ.Thus, the term “C-peptide” may encompass a single C-peptide entity or amixture of different “C-peptides”. Administration of the PEGylatedC-peptide may be by any suitable method known in the medicinal arts,including oral, parenteral, topical, or subcutaneous administration,inhalation, or the implantation of a sustained delivery device orcomposition. In one aspect, administration is by subcutaneousadministration.

Pharmaceutical compositions of the invention suitable for oraladministration may, e.g., comprise PEGylated C-peptide in sterilepurified stock powder form preferably covered by an envelope orenvelopes (enterocapsules) protecting from degradation of the drug inthe stomach and thereby enabling absorption of these substances from thegingiva or in the small intestines. The total amount of activeingredient in the composition may vary from 99.99 to 0.01 percent ofweight.

For oral administration a pharmaceutical composition comprising aPEGylated C-peptide can take the form of solutions, suspensions,tablets, pills, capsules, powders, and the like. Tablets containingvarious excipients such as sodium citrate, calcium carbonate and calciumphosphate are employed along with various disintegrants such as starchand preferably potato or tapioca starch and certain complex silicates,together with binding agents such as polyvinylpyrrolidone, sucrose,gelatin and acacia. Additionally, lubricating agents such as magnesiumstearate, sodium lauryl sulfate and talc are often very useful fortabletting purposes. Solid compositions of a similar type are alsoemployed as fillers in soft and hard-filled gelatin capsules; preferredmaterials in this connection also include lactose or milk sugar as wellas high molecular weight polyethylene glycols. When aqueous suspensionsand/or elixirs are desired for oral administration, the compounds ofthis invention can be combined with various sweetening agents, flavoringagents, coloring agents, emulsifying agents and/or suspending agents, aswell as such diluents as water, ethanol, propylene glycol, glycerin andvarious like combinations thereof.

Pharmaceutical compositions to be used in the invention suitable forparenteral administration are typically sterile aqueous solutions and/orsuspensions of the pharmaceutically active ingredients preferably madeisotonic with the blood of the recipient. Such compositions generallycomprise excipients, salts, carbohydrates, and buffering agents(preferably to a pH of from 3 to 9), such as sodium chloride, glycerin,glucose, mannitol, sorbitol, and the like.

For some applications, pharmaceutical compositions for parenteraladministration may be suitably formulated as a sterile non-aqueoussolution or as a dried form to be used in conjunction with a suitablevehicle such as sterile, pyrogen-free water. The preparation ofparenteral formulations under sterile conditions, e.g., bylyophilization, may readily be accomplished using standardpharmaceutical techniques well-known to those skilled in the art.

Pharmaceutical compositions comprising PEGylated C-peptide for use inthe present invention may also be administered topically,(intra)dermally, or transdermally to the skin or mucosa. Pharmaceuticalcompositions for topical administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed, sustained, pulsed, controlled, targeted and programmed release.Typical formulations for this purpose include gels, hydrogels, lotions,solutions, creams, ointments, dusting powders, dressings, foams, films,skin patches, wafers, implants, sponges, fibers, bandages, andmicroemulsions. Liposomes may also be used. Typical carriers includealcohol, water, mineral oil, liquid petrolatum, white petrolatum,glycerin, polyethylene glycol, and propylene glycol. Penetrationenhancers may be incorporated—see, e.g., Finnin and Morgan: J. Pharm.Sci. 88(10): 955-958, (1999). Other means of topical administrationinclude delivery by electroporation, iontophoresis, phonophoresis,sonophoresis, and microneedle or needle-free (e.g., POWDERJECT™,BOJECT™) injection.

Pharmaceutical compositions of PEGylated C-peptide for parenteraladministration may be administered directly into the blood stream, intomuscle, or into an internal organ. Suitable means for parenteraladministration include intravenous, intra-arterial, intraperitoneal,intrathecal, intraventricular, intraurethral, intrasternal,intracranial, intramuscular, intrasynovial, and subcutaneous. Suitabledevices for parenteral administration include needle (includingmicroneedle) injectors, needle-free injectors, and infusion techniques.

Subcutaneous administration of PEGylated C-peptide will typically not beinto the same site as that most recently used for insulinadministration. In one aspect of any of the claimed methods andpharmaceutical compositions, PEGylated C-peptide is administered to theopposite side of the abdomen to the site most recently used for insulinadministration. In another aspect of any of the claimed methods andpharmaceutical compositions, PEGylated C-peptide is administered to theupper arm. In another aspect of any of the claimed methods andpharmaceutical compositions, PEGylated C-peptide is administered to theabdomen. In another aspect of any of the claimed methods andpharmaceutical compositions, PEGylated C-peptide is administered to theupper area of the buttock. In another aspect of any of the claimedmethods and pharmaceutical compositions, PEGylated C-peptide isadministered to the front of the thigh.

Formulations for parenteral administration may be formulated to beimmediate and/or sustained release. Sustained release compositionsinclude delayed, modified, pulsed, controlled, targeted and programmedrelease. Thus PEGylated C-peptide may be formulated as a suspension oras a solid, semi-solid, or thixotropic liquid for administration as animplanted depot providing sustained release. Examples of suchformulations include without limitation, drug-coated stents andsemi-solids and suspensions comprising drug-loadedpoly(DL-lactic-co-glycolic)acid (PGLA), poly(DL-lactide-co-glycolide)(PLG) or poly(lactide) (PLA) lamellar vesicles or microparticles,hydrogels (Hoffman A S: Ann. N.Y. Acad. Sci. 944: 62-73 (2001)),poly-amino acid nanoparticles systems, such as the Medusa systemdeveloped by Flamel Technologies Inc., nonaqueous gel systems such asAtrigel developed by Atrix, Inc., and SABER (Sucrose Acetate IsobutyrateExtended Release) developed by Durect Corporation, and lipid-basedsystems such as DepoFoam developed by SkyePharma.

Sustained release devices capable of delivering desired doses ofPEGylated C-peptide over extended periods of time are known in the art.For example, U.S. Pat. Nos. 5,034,229; 5,557,318; 5,110,596; 5,728,396;5,985,305; 6,113,938; 6,156,331; 6,375,978; and 6,395,292; teachosmotically-driven devices capable of delivering an active agentformulation, such as a solution or a suspension, at a desired rate overan extended period of time (i.e., a period ranging from more than oneweek up to one year or more). Other exemplary sustained release devicesinclude regulator-type pumps that provide constant flow, adjustableflow, or programmable flow of beneficial agent formulations, which areavailable from, e.g., OmniPod™ Insulin Management System (InsuletCorporation, Codman of Raynham, Mass., Medtronic of Minneapolis, Minn.,Intarcia Therapeutics of Hayward, Calif., and Tricumed MedinzintechnikGmbH of Germany). Further examples of devices are described in U.S. Pat.Nos. 6,283,949; 5,976,109; 5,836,935; and 5,511,355.

Because they can be designed to deliver a desired active agent attherapeutic levels over an extended period of time, implantable deliverysystems can advantageously provide long-term therapeutic dosing of adesired active agent without requiring frequent visits to a healthcareprovider or repetitive self-medication. Therefore, implantable deliverydevices can work to provide increased patient compliance, reducedirritation at the site of administration, fewer occupational hazards forhealthcare providers, reduced waste hazards, and increased therapeuticefficacy through enhanced dosing control.

Among other challenges, two problems must be addressed when seeking todeliver biomolecular material over an extended period of time from animplanted delivery device. First, the biomolecular material must becontained within a formulation that substantially maintains thestability of the material at elevated temperatures (i.e., 37° C. andabove) over the operational life of the device. Second, the biomolecularmaterial must be formulated in a way that allows delivery of thebiomolecular material from an implanted device into a desiredenvironment of operation over an extended period of time. This secondchallenge has proven particularly difficult where the biomolecularmaterial is included in a flowable composition that is delivered from adevice over an extended period of time at low flow rates (i.e., ≦100μL/day).

Peptide drugs such as C-peptide may degrade via one or more of severaldifferent mechanisms, including deamidation, oxidation, hydrolysis, andracemization. Significantly, water is a reactant in many of the relevantdegradation pathways. Moreover, water acts as a plasticizer andfacilitates the unfolding and irreversible aggregation of biomolecularmaterials. To work around the stability problems created by aqueousformulations of biomolecular materials, dry powder formulations ofbiomolecular materials have been created using known particle formationprocesses, such as by known lyophilization, spray drying, or desiccationtechniques. Though dry powder formulations of biomolecular material havebeen shown to provide suitable stability characteristics, it would bedesirable to provide a formulation that is not only stable over extendedperiods of time, but is also flowable and readily deliverable from animplantable delivery device.

Accordingly in one aspect of any of the claimed methods andpharmaceutical compositions, the PEGylated C-peptide is provided in anonaqueous drug formulation, and is delivered from a sustained releaseimplantable device, wherein the PEGylated C-peptide is stable for atleast two months of time at 37° C.

Representative nonaqueous formulations for PEGylated C-peptide includethose disclosed in International Publication Number WO00/45790 thatdescribes nonaqueous vehicle formulations that are formulated using atleast two of a polymer, a solvent, and a surfactant.

WO98/27962 discloses an injectable depot gel composition containing apolymer, a solvent that can dissolve the polymer and thereby form aviscous gel, a beneficial agent, and an emulsifying agent in the form ofa dispersed droplet phase in the viscous gel.

WO04089335 discloses nonaqueous vehicles that are formed using acombination of polymer and solvent that results in a vehicle that ismiscible in water. As it is used herein, the term “miscible in water”refers to a vehicle that, at a temperature range representative of achosen operational environment, can be mixed with water at allproportions without resulting in a phase separation of the polymer fromthe solvent such that a highly viscous polymer phase is formed. For thepurposes of the present invention, a “highly viscous polymer phase”refers to a polymer containing composition that exhibits a viscositythat is greater than the viscosity of the vehicle before the vehicle ismixed with water.

Accordingly in another aspect of any of the claimed methods, PEGylatedC-peptide is provided in a sustained release device comprising: areservoir having at least one drug delivery orifice, and a stablenonaqueous drug formulation. In one aspect of these methods andpharmaceutical compositions, the formulation comprises: at leastPEGylated C-peptide; and a nonaqueous, single-phase vehicle comprisingat least one polymer and at least one solvent, the vehicle beingmiscible in water, wherein the drug is insoluble in one or more vehiclecomponents and the PEGylated C-peptide formulation is stable at 37° C.for at least two months. In one aspect, the solvent is selected from thegroup consisting of glycofurol, benzyl alcohol, tetraglycol,n-methylpyrrolidone, glycerol formal, propylene glycol, and combinationsthereof.

In particular, a nonaqueous formulation is considered chemically stableif no more than about 35% of the PEGylated C-peptide is degraded bychemical pathways, such as by oxidation, deamidation, and hydrolysis,after maintenance of the formulation at 37° C. for a period of twomonths, and a formulation is considered physically stable if, under thesame conditions, no more than about 15% of the C-peptide contained inthe formulation is degraded through aggregation. A drug formulation isstable according to the present invention if at least about 65% of thePEGylated C-peptide remains physically and chemically stable after abouttwo months at 37° C.

The PEGylated C-peptide can be administered intranasally or byinhalation, typically in the form of a dry powder (either alone, as amixture, e.g., in a dry blend with lactose, or as a mixed componentparticle, e.g., mixed with phospholipids, such as phosphatidylcholine)from a dry powder inhaler, as an aerosol spray from a pressurizedcontainer, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist), or nebulizer, with or without theuse of a suitable propellant, such as 1,1,1,2-tetrafluoroethane or1,1,1,2,3,3,3-heptafluoropropane, or as nasal drops. For intranasal use,the powder may comprise a bioadhesive agent, e.g., chitosan orcyclodextrin. The pressurized container, pump, spray, atomizer, ornebulizer contains a solution or suspension of the compound(s) of theinvention comprising, e.g., ethanol, aqueous ethanol, or a suitablealternative agent for dispersing, solubilizing, or extending release ofthe active, a propellant(s) as solvent and an optional surfactant, suchas sorbitan trioleate, oleic acid, or an oligolactic acid.

Prior to use in a dry powder or suspension formulation, the drug productis micronized to a size suitable for delivery by inhalation (typicallyless than 5 μm). This may be achieved by any appropriate method, such asspiral jet milling, fluid bed jet milling, supercritical fluidprocessing to form nanoparticles, high pressure homogenization, or spraydrying.

The particle size of PEGylated C-peptide of this invention in theformulation delivered by the inhalation device is important with respectto the ability of C-peptide to make it into the lungs, and preferablyinto the lower airways or alveoli. Preferably, the PEGylated C-peptideof this invention is formulated so that at least about 10% of thePEGylated C-peptide delivered is deposited in the lung, preferably about10% to about 20%, or more. It is known that the maximum efficiency ofpulmonary deposition for mouth breathing humans is obtained withparticle sizes of about 2 pm to about 3 pm. When particle sizes areabove about 5 pm, pulmonary deposition decreases substantially. Particlesizes below about 1 pm cause pulmonary deposition to decrease, and itbecomes difficult to deliver particles with sufficient mass to betherapeutically effective. Thus, particles of the PEGylated C-peptidedelivered by inhalation have a particle size preferably less than about10 pm, more preferably in the range of about 1 pm to about 5 pm. Theformulation of the PEGylated C-peptide is selected to yield the desiredparticle size in the chosen inhalation device.

Advantageously for administration as a dry powder, a PEGylated C-peptideof this invention is prepared in a particulate form with a particle sizeof less than about 10 pm, preferably about 1 to about 5 pm. Thepreferred particle size is effective for delivery to the alveoli of thepatient's lung. Preferably, the dry powder is largely composed ofparticles produced so that a majority of the particles have a size inthe desired range. Advantageously, at least about 50% of the dry powderis made of particles having a diameter less than about 10 pm. Suchformulations can be achieved by spray drying, milling, or critical pointcondensation of a solution containing the PEGylated C-peptide of thisinvention and other desired ingredients. Other methods also suitable forgenerating particles useful in the current invention are known in theart.

The particles are usually separated from a dry powder formulation in acontainer and then transported into the lung of a patient via a carrierair stream. Typically, in current dry powder inhalers, the force forbreaking up the solid is provided solely by the patient's inhalation. Inanother type of inhaler, air flow generated by the patient's inhalationactivates an impeller motor which deagglomerates the particles.

Capsules (made, e.g., from gelatin or hydroxypropylmethylcellulose),blisters and cartridges for use in an inhaler or insufflator may beformulated to contain a powder mix of the compound of the invention, asuitable powder base such as lactose or starch and a performancemodifier such as l-leucine, mannitol, or magnesium stearate. The lactosemay be anhydrous or in the form of the monohydrate, preferably thelatter. Other suitable excipients include dextran, glucose, maltose,sorbitol, xylitol, fructose, sucrose, and trehalose.

A suitable solution formulation for use in an atomizer using electrohydrodynamics to produce a fine mist may contain from 100 μg to 200 mgof PEGylated C-peptide per actuation and the actuation volume may varyfrom 1 μL to 100 μL. A typical formulation may comprise PEGylatedC-peptide propylene glycol, sterile water, ethanol, and sodium chloride.Alternative solvents that may be used instead of propylene glycolinclude glycerol and polyethylene glycol. Suitable flavors, such asmenthol and levomenthol, or sweeteners, such as saccharin or saccharinsodium, may be added to those formulations of the invention intended forinhaled/intranasal administration. Formulations for inhaled/intranasaladministration may be formulated to be immediate and/or modified releaseusing, e.g., PGLA. Modified release formulations include delayed,sustained, pulsed, controlled, targeted, and programmed release.

In the case of dry powder inhalers and aerosols, the dosage unit isdetermined by means of a valve that delivers a metered amount. Units inaccordance with the invention are typically arranged to administer ametered dose or “puff” containing from 1 mg to 200 mg of PEGylatedC-peptide. The overall daily dose will typically be in the range 1 mg to200 mg that may be administered in a single dose or, more usually, asdivided doses throughout the day.

Examples of commercially available inhalation devices suitable for thepractice of the invention are sold under the trademarks TURBHALER™(Astra), ROTAHALER® (Glaxo), DISKUS®, SPIROS™ inhaler (Dura), devicesmarketed by Inhale Therapeutics under the trademarks AERX™ (Aradigm),and ULTRAVENT® nebulizer (Mallinckrodt), ACORN II® nebulizer (MarquestMedical Products), VENTOLIN® metered dose inhaler (Glaxo), and theSPINHALER® powder inhaler (Fisons), and the like.

Kits are also contemplated for this invention. A typical kit wouldcomprise a container, preferably a vial, for the PEGylated C-peptideformulation comprising PEGylated C-peptide in a pharmaceuticallyacceptable formulation, and instructions, and/or a product insert orlabel. In one aspect, the instructions include a dosing regimen foradministration of said PEGylated C-peptide to an insulin-dependentpatient to reduce the risk, incidence, or severity of hypoglycemia. Inone aspect, the kit includes instructions to reduce the administrationof insulin by about 5% to about 35% when starting PEGylated C-peptidetherapy. In another aspect, the instructions include directions for thepatient to closely monitor their blood glucose levels when startingPEGylated C-peptide therapy. In another aspect, the instructions includedirections for the patient to avoid situations or circumstances thatmight predispose the patient to hypoglycemia when starting PEGylatedC-peptide therapy.

EXAMPLES

Abbreviations. The following abbreviations have been used in thespecification and examples: ACN=acetonitrile; Bzl=Bn=benzyl;DIEA=N,N-diisopropylethylamine; DMF=N,N-dimethylformamide;tBu=tert-butyl; TSTU=O—(N-succinimidyl)-1,1,3,3-tetramethyluroniumtetrafluoroborate; THF=tetrahydrofuran; EtOAc=ethyl acetate;DIPEA=DIEA=diisopropylethylamine; HOAt=1-hydroxy-7-azabenzotriazole;NMP=N-methylpyrrolidin-2-one; TEA=triethyl amine; SA=sinapinic acid;Su=1-succinimidyl=2,5-dioxo-pyrrolidin-1-yl; TFA=trifluoracetic acid;DCM=dichloromethane; DMSO=dimethyl sulphoxide; RT=room temperature;General Procedures: The following examples and general procedures referto intermediate compounds and final products identified in thespecification. Alternatively, other reactions disclosed herein orotherwise conventional will be applicable to the preparation of thecorresponding compounds of the invention. In all preparative methods,all starting materials are known or may easily be prepared from knownstarting materials. All temperatures are set forth in degrees Celsius (°C.) and unless otherwise indicated, all parts and percentages are byweight (i.e., w/w) when referring to yields and all parts are by volume(i.e., v/v) when referring to solvents and eluents.

Example 1 Preparation of Branched Chain PEGylated C-Peptides

Where R₁=Methyl, and n₁ and n₂ are about 450 to about 520.

One (1) g of human C— peptide (SEQ. ID. No. 1) (0.33 mM) was dissolvedin 25 mL DMF/water (20 mL/5 mL). The pH was adjusted to 7.8 withN-methylmorpholine (NMM). A solution of SUNBRIGHT GL2-400GS2 (NOFCorporation) approximate molecular weight 40,000 Da (1.85 g of theactivated PEG (0.04 mM) in DMF/water/ACN (25 mL/5 mL/10 mL) was thenadded and the reaction was stirred overnight at room temperature.

The solution was diluted with purified water to 700 mL. The DMFconcentration was 6% v/v. The pH was adjusted to 4 to 4.5 with aceticacid and filtered. The solution was purified by HPLC using a YMC-ODScolumn (2.5×30 cm) using an 0.5% acetic acid (mobile phase A)/100 mMsodium acetate (mobile phase B)/ACN (mobile phase C) gradient.Separations were completed by equilibrating the column with three columnvolumes of 90% A/10% C. PEGylated C-peptide was loaded on to, and washedwith, 90% B/10% C (three column volumes), followed by isocratic washingwith one column volume of 90% A/10% C. Elution was achieved via amulti-linear gradient starting with 90% A/10% C to 70% A/30% C over twocolumn volumes, followed by a linear gradient consisting of 70% A/30% Cand rising to 50% A/50% C over five column volumes.

The pool from the HPLC (140 mL) was diluted with 70 mL purified waterand evaporated to a volume of 130 mL at 25° C. The final concentrationwas 12 g/L. The solution was lyophilized to yield 1500 mg of PEGylatedC-peptide (80% yield).

Fractions collected after purification were analyzed by reverse phaseand size exclusion HPC. Reverse phase HPLC was conducted using a WatersHPLCBEH C18 17 μM column, using a mobile phase of ACN and 0.1% TFA usinga two component linear gradient of 24% to 40% ACN over 3 minutes,followed by 40% to 90% ACN over 1 minute, at a flow rate of 0.25 mL/min,a column temperature of 40° C., and a sample concentration of 5 mg/mL. Arepresentative chromatogram is presented in FIG. 1.

Size exclusion chromatography was conducted using a Superdex 75,10/300GL column using an isocratic elution with a mobile phase of 0.1 Mphosphate buffer pH 7.4 containing 2.7 mM KCl and 0.137 M NaCl at a flowrate of 0.5 mL/min. A representative chromatogram is presented in FIG.2.

Example 2 Preparation of Additional Branched Chain PEGylated C-Peptides

Using similar reaction conditions as described for Example 1, and usingthe following reagents in the place of SUNBRIGHT GL2-400GS2, thefollowing PEGylated C-peptides of MW 10 kDa to 80 kDa may be readilyprepared.

Use of SUNBRIGHT GL2-400TS yields:

Where R₁=Methyl.

Use of SUNBRIGHT GL3-400TS100U yields:

Where R₁=Methyl.

Use of SUNBRIGHT GL3-400GS100U yields:

Where R₁=Methyl.

Use of SUNBRIGHT GL3-400HS100U yields:

Where R₁=Methyl.

Use of SUNBRIGHT LY-400NS yields:

Where R₁=Methyl.

Use of the active intermediate

yields:

Where R₁=methyl.

Example 3 Preparation of Linear Chain PEGylated C-Peptides

Using similar reaction conditions as described for Example 1, and usingthe following reagents in the place of SUNBRIGHT GL2-400GS2, thefollowing PEGylated C-peptides of MW 5 kDa to 80 kDa may be readilyprepared.

SUNBRIGHT ME-200GS.

Fractions collected after purification were analyzed by reverse phaseand size exclusion HPC. Reverse phase HPLC was conducted using a WatersHPLCBEH C18 17 μM column, using a mobile phase of ACN and 0.1% TFA usinga two component linear gradient of 24% to 40% ACN over 3 minutes,followed by 40% to 90% ACN over 1 minute, at a flow rate of 0.25 mL/min,a column temperature of 40° C. and a sample concentration of 5 mg/mL. Arepresentative chromatogram is presented in FIG. 3.

Size exclusion chromatography was conducted using a Superdex 75,10/300GL column using an isocratic elution with a mobile phase of 0.1 Mphosphate buffer pH 7.4 containing 2.7 mM KCl and 0.137 M NaCl at a flowrate of 0.5 mL/min. A representative chromatogram is presented in FIG.4.

Example 4 Preparation of Additional Linear Chain PEGylated C-Peptides

Using similar reaction conditions as described for Example 1, and usingthe following reagents in the place of SUNBRIGHT GL2-400GS2, thefollowing linear PEGylated C-peptides of MW 5 kDa to 80 kDa may bereadily prepared.

Use of SUNBRIGHT ME-200CS yields:

Use of SUNBRIGHT ME-200HS yields:

Use of SUNBRIGHT ME-200TS yields:

Use of SUNBIO P1PAL-30 yields:

Use of SUNBIO P1APAL-30 yields:

Use of SUNBIO P1TPAL-5 yields:

Use of SUNBIO P1BAL-30 yields:

Use of SUNBIO P1ABAL-30 yields:

Use of SUNBIO P1TBAL-5 yields:

Example 5 Measurement of Pharmacokinetic Characteristics in Dogs

A pharmacokinetic (PK) study was conducted to determine the C-peptide PKprofile with unmodified C-peptide in beagle dogs.

Methods: Two male and one female dog received S.C. the unmodifiedsynthetic human C-peptide (0.5 mg/kg; 0.025 mL/kg) formulated inphosphate buffered saline (20 mg/mL). Dogs were bled by venipuncture andblood samples were collected at predetermined time points over 14 days.Plasma samples were obtained after centrifugation of the blood (3,000rpm for 10 minutes) and stored at −80° C. until analysis. A CRO withGood Laboratory Practice (GLP) capabilities (MicroConstants, Inc.; SanDiego, Calif.) performed the bioanalytical work. Plasma levels ofC-peptide were measured by an enzyme-linked immunosorbant assay (ELISA)technique based on a commercial kit for human C-peptide determination(Mercodia; catalog number 10-1136-01) using the manufacturer'sinstructions. Results were expressed as C-peptide concentrations. Forthe PK analysis, the below quantitation level (BQL) was treated as zeroand nominal time points were used for all calculations. PK parameterswere determined by standard model independent methods based on theindividual plasma concentration-time data for each animal using model200 in WinNonlin Professional 5.2.1 (Pharsight Corp., Mountain View,Calif.)

Results: All animals survived the duration of the study. Each treatmentwas well tolerated based on the absence of clinical abnormalities. Themean±standard deviation (SD) for C-peptide maximum concentration(C_(max)) and area under the curve (AUC_((0-t))) values following S.C.dosing of the unmodified C-peptide in dogs are presented in Table E1below. The corresponding mean±SD C-peptide plasma concentration-timeprofiles on a linear scale after 1 day and 12 days post dose arepresented in FIG. 5A and FIG. 5B, respectively. Single-doseadministration of unmodified C-peptide resulted in a rapid peakaccumulation, and then rapid loss of C-peptide from the circulation indogs. The use of unmodified C-peptide resulted in circulating levels ofC-peptide that were BQL within half a day.

TABLE E1 Mean PK Parameters of C-Peptide in Dogs Following a Single S.C.Dose of Unmodified Aqueous C-peptide (CP-AQ) C_(max) (ng/mL) AUC_((0-t))(ng · day/mL)^(a) Mean SD Mean SD CP-AQ 757 192 77.4 6.82^(a)AUC_((0-t)) is the area under the plasma concentration-time curvefrom immediate post dose to the last measurable sampling time and iscalculated by the linear trapezoidal rule.

A second PK study was conducted to determine the C-peptide PK profilesusing two representative PEGylated C-peptides of different molecularweights (a 20 kDa linear PEG (Example 3) and a 40 kDa branched PEG(Example 1)) in beagle dogs. Results were compared to those forunmodified C-peptide.

Methods: Three male dogs received S.C. the 20 kDa PEGylated synthetichuman C-peptide (0.5 mg/kg equivalents of C-peptide; 0.09 mL/kg)formulated in phosphate buffered saline (50.8 mg/mL PEGylated C-peptide)and three male dogs received S.C. the 40 kDa PEGylated synthetic humanC-peptide (0.5 mg/kg equivalents of C-peptide; 0.012 mL/kg) formulatedin phosphate buffered saline (82.5 mg/mL PEGylated C-peptide). Dogs werebled by venipuncture and blood samples were collected at predeterminedtime points over 21 days. Plasma samples were obtained aftercentrifugation of the blood (3,000 rpm for 10 minutes) and stored at−80° C. until analysis. A CRO with GLP capabilities (MicroConstants,Inc.; San Diego, Calif.) performed the bioanalytical work. Plasma levelsof C-peptide were measured by an ELISA technique based on a commercialkit for human C-peptide determination (Mercodia; catalog number10-1136-01) using PEGylated C-peptide quality control standards. Resultswere expressed as C-peptide concentrations. For the PK analysis, the BQLwas treated as zero and nominal time points were used for allcalculations. PK parameters were determined by standard modelindependent methods based on the individual plasma concentration-timedata for each animal using model 200 in WinNonlin Professional 5.2.1(Pharsight Corp., Mountain View, Calif.).

Results: All animals survived the duration of the study. Each treatmentwas well tolerated based on the absence of clinical abnormalities. Bycomparison to unmodified C-peptide, the use of the PEGylated C-peptideextended C-peptide exposure in the dog to at least 4 days post dose withthe 20 kDa linear PEGylated C-peptide, and to at least 15 days post dosewith the 40 kDa branched chain PEGylated C-peptide.

The mean±SD for C-peptide C_(max), AUC_((0-t)), and half-life (T_(1/2))values following S.C. dosing of the 20 kDa PEGylated C-peptide and the40 kDa PEGylated C-peptide in dogs are presented in Table E2 below. Thecorresponding mean±SD C-peptide plasma concentration-time profiles onlinear and semi-logarithmic scales are presented in FIG. 6 and FIG. 7,respectively.

TABLE E2 Mean PK Parameters of C-Peptide in Dogs Following a Single S.C.Dose of a 20 kDa and a 40 kDa PEGylated C-peptide (CP) Ratio of PEG (20PEG (40 PEG (40 kDa) kDa) CP kDa) CP CP to PEG Mean SD Mean SD (20 kDa)CP C_(max) 1,800 400 5,790 642 2.26 (ng/mL) AUC_((0-t)) 4,060 588 38,6008,200 6.67 (ng · day/mL)^(a) T_(1/2) ^(b) 1.39 0.197 2.02 0.217 1.45(Day) ^(a)AUC_((0-t)) is the area under the plasma concentration-timecurve from immediate post dose to the last measurable sampling time andis calculated by the linear trapezoidal rule. ^(b)T_(1/2) is theterminal half-life calculated by In(2)/λ where λ represents theelimination rate constant for the log-linear portion of the terminalphase.

Surprisingly, the C_(max) and AUC_((0-t)) values following S.C. dosingof the 40 kDa PEGylated C-peptide were 2.26- and 6.67-fold higher thanthe corresponding values of the kDa PEGylated C-peptide, respectively.Thus, the 40 kDa branched chain PEGylated C-peptide provides forsignificantly improved PK properties compared to the 20 kDa PEGylatedC-peptide, or compared to unmodified C-peptide. A third PK study wasconducted in beagle dogs with lower doses of the 40 kDa branched chainPEGylated C-peptide (0.006, 0.025, and 0.1 mg/kg, via single-dose S.C.injection of PEGylated synthetic human C-peptide (40 kDa PEG; made viaSUNBRIGHT GL2-400GS2, Example 1)) formulated in phosphate bufferedsaline.

Methods: Plasma levels of C-peptide were measured over 14 days. Anonclinical CRO (Bio-Quant, Inc.; San Diego, Calif.) performed theanimal portion of the study. Nine male and female dogs weighedapproximately 7-12 kg and 6-8 kg, respectively, on the day of doseadministration (Day 0). Animals were fed during the study and foodconsumption was determined on Days 0, 1, and 2. Body weights were alsodetermined on Days 7 and 14. The injection area on the back of eachanimal was shaved and cleaned two days prior to Day 0. There were threegroups of dogs (n=2 males and 1 female/group):

Group 1 received a single S.C. injection via 25 G needle of 2 mg/mLPEGylated C-peptide at a dose of 0.05 mL/kg (containing 0.005 mg/kgequivalents of C-peptide).

Group 2 received a single S.C. injection via 25 G needle of 2 mg/mLPEGylated C-peptide at a dose of 0.0125 mL/kg (containing 0.00125 mg/kgequivalents of C-peptide).

Group 3 received a single S.C. injection via 25 G needle of 0.4 mg/mLPEGylated C-peptide at a dose of 0.015 mL/kg (containing 0.0003 mg/kgequivalents of C-peptide).

Dogs were bled by venipuncture and blood samples were collected at Day 0(pre-dose, 30 minutes, 1 hour, 3 hour, and 6 hour) and Days 1, 2, 3, 4,7, 10, and 14. Plasma samples were obtained after centrifugation of theblood (3,000 rpm for 10 minutes) and stored at −80° C. until analysis. ACRO with GLP capabilities (MicroConstants, Inc.; San Diego, Calif.)performed the bioanalytical work. Plasma levels of C-peptide weremeasured by an ELISA technique based on a commercial kit for humanC-peptide determination (Mercodia; catalog number 10-1136-01) usingPEGylated C-peptide quality control standards. Results were expressed asC-peptide concentrations. For the PK analysis, the BQL was treated aszero and nominal time points were used for all calculations. PKparameters were determined by standard model independent methods basedon the individual plasma concentration-time data for each animal usingmodel 200 in WinNonlin Professional 5.2.1 (Pharsight Corp., MountainView, Calif.).

Results: All animals survived the duration of the study. Each treatmentwas well tolerated based on the absence of clinical abnormalities. Themean±SD for C-peptide C_(max), AUC_((0-t)), and T_(1/2) values followingsingle S.C. doses of PEGylated C-peptide in dogs are presented in TableE3. The corresponding mean±SD for C-peptide plasma concentration-timeprofiles on linear and semi-logarithmic scales are presented in FIG. 8Aand FIG. 8B, respectively. The mean±SD for C-peptide C_(max) andAUC_((0-t)) values are presented in FIG. 9A and FIG. 9B, respectively.

TABLE E3 Pharmacokinetic Parameters of C-Peptide in Dogs FollowingSingle S.C. Doses of PEGylated C-peptide (40 kDa PEG) PEGylatedC-peptide Dose (mg/kg equivalents of C-peptide) 0.005 0.00125 0.0003Mean SD Mean SD Mean SD Cmax (ng/mL) 73.2 2.23 19.0 4.00 1.74 1.53AUC(0-t) 470 84.3 91.9 8.86 4.17 3.94 (ng · day/mL) a T½ b (day) 2.440.599 1.93 0.530     1.76 c ND a AUC(0-t) is the area under the plasmaconcentration-time curve from immediate post dose to the last measurablesampling time and is calculated by the linear trapezoidal rule. b T½ isthe terminal half-life calculated by ln(2)/λ where λ represents theelimination rate constant for the log-linear portion of the terminalphase. c n = 1 and SD was ND (not determined)

In summary, following single S.C. escalating doses of 40 kDa PEGylatedC-peptide in dogs, exposure of C-peptide is significantly increased. Theresults from the second and third PK studies in dogs confirm that the 40kDa branched chain PEGylated C-peptide provides for significantlyimproved PK properties compared to the 20 kDa linear chain PEGylatedC-peptide, or comparison to unmodified C-peptide (first PK study indogs). Furthermore, the 40 kDa branched chain PEGylated demonstrated nosignificant adverse side effects at the doses tested.

Example 7 Pharmacokinetics in Sprague Dawley Rats Following Single doses.c. Administration

The PK of the 40 kDa PEGylated C-peptide (Example 12) was assessed inSprague Dawley rats (2/sex/group) following single-dose s.c.administration of 0.0413, 0.167, and 0.664 mg/kg. Blood samples werecollected prior to and for 14 days after administration. Plasma sampleswere analyzed for PEGylated C-peptide using the ELISA method, asdescribed above for the dog study. Individual PK parameters wereestimated using “non-compartmental” methods. The mean (±SD) plasmaconcentration-time profiles following single-dose s.c. administrationare illustrated in FIG. 10 with the corresponding PK parameterssummarized in Table E4.

TABLE E4 PEGylated C-peptide pharmacokinetics in Sprague Dawley ratsfollowing single-dose subcutaneous administration Pharmaco- CBX129801Dose, mg/kg kinetic 0.0413 0.167 0.664 Parameter^(a) Mean SD Mean SDMean SD T_(1/2), day 1.35 0.186 1.27 0.109 1.27 0.175 T_(max), day 1.000.00 1.00 0.00 1.00 0.00 C_(max), nM 3.38 0.869 12.3 2.61 34.1 10.4AUC₍₀₋₇₎, 8.13 1.45 33.0 7.27 101 18.7 nM · day AUC₍₀₋₇₎, 374 66.6 1,520334 4,660 862 ng · day/ mL AUC_((0-t)), 8.13 1.45 33.9 8.31 107 19.6 nM· day AUC_((0-t)), 374 66.6 1,560 382 4,920 900 ng · day/ mLAUC_((0-inf)), 8.48 1.43 34.6 7.88 108 19.4 nM · day AUC_((0-inf)), 39065.7 1,590 362 4,950 893 ng · day/ mL CL/F, 109 20.5 109 25.5 138 25.5mL/day/kg Vd/F, 214 62.0 201 50.6 250 42.9 mL/kg ^(a)Refer to List ofAbbreviations (Table A) for definition of terms.

Example 8 Pharmacokinetics in Cynomolgus Monkeys Following Single Doses.c. Administration

The PK of the 40 kDa PEGylated C-peptide (Example 12) was assessed inCynomolgus monkeys (2/sex) following single-dose s.c. administration of0.0827 mg/kg. Blood samples were collected prior to and for 14 daysafter administration. Plasma samples were analyzed for CBX129801 usingthe ELISA method as described above for the dog study. Individual PKparameters were estimated using “non-compartmental” methods. The mean(±SD) plasma concentration-time profile following single-dose s.c.administration is illustrated in FIG. 11 with the corresponding PKparameters summarized in Table E5.

TABLE E5 Pharmacokinetic Parameters of PEGylated C-peptide in CynomolgusMonkeys, at 0.0827 mg/kg Pharmacokinetic Parameter Mean SD T_(max), day2.00 0.816 T_(1/2), day 5.44 2.05 C_(max), nM 13.2 1.32 AUC₍₀₋₇₎, nM ·day 67.5 7.01 AUC₍₀₋₇₎, ng · day/mL 3,110 323 AUC₍₀₋₁₄₎, nM · day 95.716.9 AUC₍₀₋₁₄₎, ng · day/mL 4,400 770 AUC_((0-inf)), nM · day 121 37.0AUC_((0-inf)), ng · day/mL 5,560 1,710 CL/F, mL/day/kg 16.0 4.90 Vd/F,mL/kg 115 10.0 ^(a) Refer to List of Abbreviations (Table A) fordefinition of terms.

These results indicate that peak concentration occurred within 2 days;the T_(1/2) is ˜5.4 days. Since the monkeys were not fasted and becausethe detection antibody cross-reacts with monkey C-peptide, the resultsfor the 40 kDa PEGylated C-peptide (Example 12) include endogenousC-peptide levels. Therefore at the later time points (i.e., Days 10 and14) when the measured PEGylated C-peptide levels were lower, thecontribution of endogenous monkey C-peptide could have confounded theresults. With the Day 14 time point removed from the analysis, theT_(1/2) is 3.7 days. In summary for these single-dose PK studies,following s.c. administration, the peak PEGylated C-peptide plasmaconcentration generally occurs within 1 to 5 days. AUC and C_(max)increase with increasing dose and are generally dose proportional.

Example 9 Repeat-Dose Pharmacokinetic Studies with Unmodified C-Peptide

In the five GLP toxicology studies conducted with non-PEGylatedC-peptide for up to 4 weeks in rats and 13 weeks in Cynomolgus monkeys,the C-peptide was continuously infused s.c. via implanted osmotic pumps.The plasma concentration of C-peptide was measured periodicallythroughout the studies and a steady-state concentration (C_(ss)) overthe duration of exposure has been determined for each study as shown inTable E6.

TABLE E6 Summary of unmodified C-peptide concentrations in repeated-dose toxicity studies at the no observed effect level No Observed EffectC-peptide Species Duration Level C_(ss) ^(a)(nM) Sprague Dawley 14 days2 mg/kg/day 27^(b) rat Sprague Dawley 14 days 2 mg/kg/day 16^(b) ratSprague Dawley 4 weeks 0.5 mg/kg/day   4.2^(c) rat Cynomolgus 14 days 2mg/kg/day 84^(b) monkey Cynomolgus 13 weeks 3.6 mg/kg/day 40^(d) monkey^(a)Test article delivered by continuous s.c. infusion. ^(b)Mean C_(ss)estimated from the C-peptide plasma levels on days 2, 10, and 14.^(c)Mean C_(ss) estimated from the C-peptide plasma levels on days 2,14, and 28. ^(d)Mean C_(ss) estimated from the C-peptide plasma levelson days 2, 28, 37/38, 56, and 91

In conclusion, there were sustained C-peptide levels throughout theduration of dosing in these toxicology studies.

Example 10 Repeat-Dose Pharmacokinetic Studies

The PK of the 40 kDa PEGylated C-peptide (Example 12) has been assessedfollowing multiple dose administration in rats and monkeys. Thesestudies are summarized below.

Pharmacokinetics in Sprague Dawley Rats Following Repeated-DoseSubcutaneous Administration (Weekly) for 28 Days.

Methods: The PK of the 40 kDa PEGylated C-peptide (Example 12) wasassessed in Sprague Dawley rats (3/sex/group per time point) followingmultiple dose subcutaneous administration of 2.74, 8.22, and 27.4mg/kg/week for 5 doses. Blood samples were collected prior to and for 7days following the first dose. Trough blood samples were collected afterthe 2nd through 4th doses. Following the last dose (5th dose), bloodsamples were collected for 28 days. Plasma samples were analyzed forPEGylated C-peptide using an ELISA method, as described previously. PKparameters were estimated using “non-compartmental” methods and the meanconcentration-time profile for each dose by gender.

Results: The mean (±SD) plasma concentration-time profiles upon multipledose s.c. administration by gender are illustrated in FIG. 12 with thecorresponding PK parameters summarized in Table E7. The relationshipsbetween dose and the primary

TABLE E7 Summary of Pharmacokinetic Characteristics in Sprague DawleyRats Following Repeated-Dose Subcutaneous Administration (Weekly) for 28Days Sex Male Female Dose, mg/kg/week 2.74 8.22 27.4 2.74 8.22 27.4First-Dose Parameters^(a) C_(max), nM 156 440 1,540 412 564 2,210T_(max), day^(b) 1.00 1.00 2.00 3.00 1.00 1.00 T_(1/2), day 1.52 1.491.62 1.47 1.26 1.46 AUC_(tau), nM · day 507 1,580 4,580 1,020 2,1107,590 AUC_(inf), nM · day 539 1,690 4,950 1,060 2,190 8,050 CL/F,mL/day/kg 108 103 118 55.1 79.7 72.5 V_(d)/F, mL/kg 237 222 275 117 145153 C_(max) 0.379 0.780 0.697 — — — Male/Female Ratio AUC_(inf) 0.5080.772 0.615 — — — Male/Female Ratio CL/F 1.96 1.30 1.63 — — —Male/Female Ratio V_(d)/F 2.03 1.53 1.80 — — — Male/Female RatioRepeated-Dose Parameters^(a) C_(max), nM 147 438 1,070 311 846 2,660T_(max), day^(b) 2.00 1.00 2.00 1.00 1.00 2.00 T_(1/2), day 1.43 1.511.66 1.65 1.35 1.84 AUC_(tau), nM · day 524 1,710 4,150 1,090 3,0009,590 CLss/F, mL/day/kg 111 102 141 53.6 58.3 60.9 V_(d)ss/F, mL/kg 230223 337 128 114 161 C_(max) 0.473 0.518 0.402 — — — Male/Female RatioAUC_(tau) 0.481 0.570 0.433 — — — Male/Female Ratio CLss/F 2.07 1.752.32 — — — Male/Female Ratio V_(d)ss/F 1.80 1.96 2.09 — — — Male/FemaleRatio Repeated/1st 0.942 0.995 0.695 0.755 1.50 1.20 Dose C_(max) RatioRepeated/1st 1.03 1.08 0.906 1.07 1.42 1.26 Dose AUC_(tau) Ratio^(a)Refer to List of Abbreviations (Table A) for definition of terms;^(b)Expressed as median.

Overall, these results indicate that steady state is achieved by Day 14to Day 21. Exposure increased with increasing dose with peak plasmaconcentrations occurring between Day 1 and Day 3. AUC and C_(max)following the first and last dose appear to be dose proportional overthe range assessed with AUC at steady state similar to that followingthe first dose (AUC steady-state to first dose ratio ranging from 0.7 to1.5). The T₁₂ appears similar across the 3 doses and following the firstand last dose (˜1.5 days). Systemic exposure in females is ˜2 foldhigher than males. Detectable drug levels persisted for approximately 2weeks at all doses in the 28-day recovery period following cessation ofdosing with no detectable drug at the end of the recovery period exceptin one male (2.74 mg/kg/week) and one female (8.22 mg/kg/week). Due toinitial technical difficulties with the anti-drug antibody (ADA) assay,results are not available for anti-PEGylated C-peptide antibodies. Thehigh exposures, generally dose proportional results, and consistentclearance between the first and the last doses with PEGylated C-peptidesupports the proposition that any antibody formation did not likelyimpact exposure to any significant degree.

Pharmacokinetics in Cynomolgus Monkeys Following Repeated-DoseSubcutaneous Administration (Weekly) for 28 Days.

Methods: The PK of the 40 kDa PEGylated C-peptide (Example 12) wasassessed in Cynomolgus monkeys (5/sex/group) upon multiple dose s.c.administration of 1.33, 4.0, and 13.3 mg/kg/week for 5 doses. Bloodsamples were collected prior to and for 7 days following the first dose.Trough blood samples were collected after the 2nd through 4th doses.Following the last dose (5^(th) dose), blood samples were collected forup to 28 days. Plasma samples were analyzed for PEGylated C-peptideusing an ELISA method, as described previously. PK parameters wereestimated using “non-compartmental” methods.

Results: The mean plasma concentration-time profiles upon multiple doses.c. administration by gender are illustrated in FIG. 14 with thecorresponding PK parameters summarized in Table E8. The relationshipsbetween dose and the primary parameters of exposure (AUC and C_(max))are shown FIG. 15.

TABLE E8 Summary of Pharmacokinetic Characteristics in CynomolgusMonkeys Following Repeated-Dose Subcutaneous Administration (Weekly) for28 Days Male Female Pooled Gender Dose, mg/kg 1.33 4.0 13.3 1.33 4.013.3 1.33 4.0 13.3 First-Dose Parameters^(a) C_(max), nM 250 881 2,780252 877 2,400 251 879 2,590 T_(max), day^(b) 2.00 1.00 2.00 2.00 2.002.00 2.00 1.50 2.00 T_(1/2), day 4.67 3.30 2.85 3.33 3.53 2.63 4.00 3.422.74 AUC_(tau), nM · day 1,180 4,210 13,200 1,280 3,920 11,200 1,2304,060 12,200 AUC_(inf), nM · day 1,920 5,770 17,700 1,800 5,410 14,4001,860 5,590 16,000 CL/F, mL/day/kg 15.3 15.5 16.9 16.1 16.5 21.0 15.716.0 19.0 V_(d)/F, mL/kg 101 72.7 68.0 77.4 81.7 79.8 89.0 77.2 73.9C_(max) M/F Ratio 0.922 1.00 1.16 — — — — — — AUC_(tau) M/F Ratio 0.9221.07 1.18 — — — — — — Repeated-Dose Parameters^(a) C_(max), nM 308 1,2503,780 371 1,350 4,900 340 1,300 4,340 T_(max), day^(b) 2.00 1.00 2.002.00 1.00 2.00 2.00 1.00 2.00 T_(1/2), day 4.78 2.93 3.91 4.62 3.34 3.074.70 3.17 3.49 AUC_(tau), nM · day 1,650 5,610 18,600 1,860 6,000 21,4001,750 5,810 20,000 AUC_(inf), nM · day 2,740 7,330 28,300 2,910 8,24028,300 2,820 7,790 28,300 CLss/F, mL/day/kg 18.3 15.7 15.8 15.9 14.713.6 17.1 15.2 14.7 V_(d)ss/F, mL/kg 121 67.8 86.0 110 71.4 61.0 11669.6 73.5 C_(max) M/F Ratio 0.830 0.926 0.771 — — — — — — AUC_(tau) M/FRatio 0.887 0.935 0.869 — — — — — — Repeated/First Dose 1.23 1.42 1.361.47 1.54 2.04 1.35 1.48 1.68 C_(max) Ratio Repeated/First Dose 1.401.33 1.41 1.45 1.53 1.91 1.42 1.43 1.64 AUC_(tau) Ratio ^(a)Refer toList of Abbreviations (Table A) for definition of terms; ^(b)Expressedas median.

Overall, these results indicate that steady state is achieved byapproximately Day 14. Exposure increased with increasing dose with peakplasma concentrations occurring between Day 1 and Day 2. AUC and C_(max)following the first and last dose appear to be dose proportional overthe range assessed with AUC_(tau) at steady state approximately 30% to90% higher than observed following the first dose. The T_(1/2) appearssimilar across the 3 doses and following the first and last dose (˜3days). Systemic exposure in females was similar to that observed inmales. Detectable drug levels persisted at both recovery doses (4.0 and13.3 mg/kg/week) following cessation of dosing; however, the plasmaconcentrations of the PEGylated C-peptide markedly decreased over timeand were substantially lower by the end of the recovery period for alldoses (approximately 2-3 times the lower limit of quantitation). At theend of the recovery period, there was a modest ADA response at the lowertwo doses and a strong response at the high dose; however, assignificant drug levels were present throughout and well aftercompletion of dosing, the presence of antibodies did not meaningfullyimpact the monkeys' exposure to the PEGylated C-peptide.

Example 11 Effect on Nerve Conduction Velocity (NCV) in STZ InducedDiabetic Rats

To assess the effect of the PEGylated C-peptide on nerve conductionvelocity in diabetic rats, the 40 kDa branched PEG (Example 12)) wasadministered to STZ induced diabetic rats for 8 weeks. Results were alsocompared to those for unmodified human C-peptide. PEGylated ratC-peptide, which was coupled to the same 40 kDa branched PEG asdescribed in Example 1, and unmodified rat C-peptide.

Protocols and Methods: Streptozotocin (STZ) was administered I.V. at adose of 50 mg/kg via the injection of 1 ml of a 50 mg/mL standardsolution of STZ. Sprague Dawley male rats were obtained from Harlan.Rats had an average weight of around 400 g, fed a standard diet (TD2014)and housed individually in standard solid bottom 8-inch deep plasticswith corn cob bedding. Animals were housed with a normal, 12 hourslight, 12 hours dark light cycle and at an average temperature of 72±8°F. and relative humidity of: 30%-70% for the duration of the study.Animals were dosed for a period of 8 weeks, according to the dosing andformulations listed in Table E9.

TABLE E9 Summary of dosing protocols Dose STZ Dose Dose Volume # of GrpCompound 50 mg/kg mg/kg ml/kg Route Freq An's An #'s 1 Vehicle No 0 1 SC1/wk** 5 1-5 2 Vehicle Yes 0 1 SC 1/wk** 10  6-15 3 PEGylated Yes 1 1 SC1/wk** 10 16-25 human C- peptide 4 PEGylated Yes 3 1 SC 1/wk** 10 26-35human C- peptide 5 Un modified Yes 1.5* pump pump Twice 10 36-45 HumanC- peptide 6 Un modified Yes 0.3* pump pump Twice 10 46-55 Rat C-peptide7 PEGylated Yes 0.3 1 SC 1/wk** 10 56-65 rat C-peptide TOTAL 65 ANIMALS*Per 24 hours; **2/week in the first week of dosing.

The required dose of each drug administered to each animal wascalculated based on the most recent body weight. Sterilephosphate-buffered saline was used as the vehicle.

Pretreatment Phase study conduct: Prior to starting treatment animalswere observed to identify any abnormalities, signs of pain or distressand any findings recorded, were discussed with a clinical veterinarianwhen observed. Body weights were determined before STZ treatment (day1), for randomization to treatment groups, on day 7, and 11 and onceweekly thereafter. Food Weights were determined pre-STZ (day 1), atrandomization to treatment groups, and on days 7, and 11 and once weeklythereafter. Animals were randomized for the treatment phase based onC-peptide (<0.4 nM), whole blood glucose values (400-600 mg/dL) and bodyweight values. (See Table E5) Randomization was achieved using B.R.A.T.(block randomization allocation tool). Subcutaneous pump implants (Alzetpumps model 2ML4) were surgically implanted on day 10 and day 39.

Treatment Phase study conduct: Blood was collected via a tail bleed onday 3 for randomization, day 7, day 11 and weekly thereafter for glucoseand/or C-peptide. Animals were fasted for 6 hours prior to STZinjection, 3 hours prior to every glucose evaluation and fed ad lib forthe remainder of the study. (See Table E10).

TABLE E10 Study Schedule Weekday Day Task Monday ~−7 Animals arriveMonday 1 6 hour fast, body and food weights, IV administration of STZ(Zanosar) Wednesday 3 3 hour fast (6:00 am) prior to blood collection.Body and food weights. Tail bleed (9:00 am) for whole blood glucoseevaluation in duplicate using glucometer and for EDTA plasma C-peptideanalysis Sunday 7 3 hour fast (6:00 am) prior to blood collection. Bodyand food weights. Tail bleed (9:00 am) for whole blood glucoseevaluation in duplicate using glucometer and for EDTA plasma C-peptideanalysis Monday 8 Randomize to treatment groups (am). Baseline NCVmeasurements (pm) Tuesday 9 Baseline NCV measurements Wednesday 10Implantation of pumps and twice per week SC dosing (8:00 am) beginsThursday 11 3 hour fast (6:00 am) prior to blood collection. Body andfood weights. Tail bleed (9:00 am) for whole blood glucose evaluation induplicate using glucometer and for EDTA plasma C-peptide analysis(GROUPS 3, 4 and 7. The first five animals will be bleed 24 hourspostdose, the second five will be bleed 48 hours postdose on day 12)Friday 12 Blood collection for EDTA plasma on the second five animalsfrom groups 3, 4 and 7 - 48 hour post dose collection. Saturday 13 SCdosing (8:00 am) Wednesday 17 3 hour fast (6:00 am) prior to bloodcollection. Body and food weights. SC dosing (8:00 am). Tail bleed (9:00am) for whole blood glucose evaluation in duplicate using glucometer andfor EDTA plasma C-peptide analysis Wednesday 24 3 hour fast (6:00 am)prior to blood collection. Body and food weights. SC dosing (8:00 am).Tail bleed (9:00 am) for whole blood glucose evaluation in duplicateusing glucometer and for EDTA plasma C-peptide analysis (GROUPS 3, 4 and7. The first five animals will be bleed 24 hours postdose, the secondfive will be bleed 48 hours postdose) Thursday 25 Blood collection forEDTA plasma on the first five animals from groups 3, 4 and 7 - 24 hourpost dose collection. Friday 26 Blood collection for EDTA plasma on thesecond five animals from groups 3, 4 and 7 - 48 hour post dosecollection. Saturday 27 SC dosing (8:00 am) Wednesday 31 3 hour fast(6:00 am) prior to blood collection. Body and food weights. SC dosing(8:00 am). Tail bleed (9:00 am) for whole blood glucose evaluation induplicate using glucometer. Saturday 34 SC dosing (8:00 am) Monday 36 3hour fast (6:00 am) prior to blood collection. Body and food weights. SCdosing (8:00 am). Tail bleed (9:00 am) for whole blood glucoseevaluation in duplicate using glucometer and for EDTA plasma C-peptideanalysis Tuesday 37 NCV measurements Wednesday 38 SC dosing (8:00 am),NCV measurements Thursday 39 Removals of old pumps and implant newpumps. Saturday 41 SC dosing (8:00 am) Wednesday 45 3 hour fast (6:00am) prior to blood collection. Body and food weights. SC dosing (8:00am). Tail bleed (9:00 am) for whole blood glucose evaluation induplicate using glucometer Saturday 48 SC dosing (8:00 am) Wednesday 523 hour fast (6:00 am) prior to blood collection. Body and food weights.SC dosing (8:00 am). Tail bleed (9:00 am) for whole blood glucoseevaluation in duplicate using glucometer Saturday 55 SC dosing (8:00 am)Wednesday 59 3 hour fast (6:00 am) prior to blood collection. Body andfood weights. SC dosing (8:00 am). Tail bleed (9:00 am) for whole bloodglucose evaluation in duplicate using glucometer Saturday 62 SC dosing(8:00 am) Monday 64 NCV measurements Tuesday 65 NCV measurementsWednesday 66 3 hour fast (6:00 am) prior to blood collection. Body andfood weights. SC dosing (8:00 am). Tail bleed (9:00 am) for whole bloodglucose evaluation in duplicate using glucometer and for EDTA plasmaC-peptide analysis Kidney Necropsy in the afternoon.

Electrophysiologic Endpoints: Digital Nerve Action Potentials wererecorded with the active recording electrode positioned at the ankle,behind the lateral malleolus and the stimulating cathode at the base ofthe second digit of the hindpaw. Velocity was calculated by dividing thedistance between the stimulating cathode and the active electrode by theabsolute onset latency of the initial depolarizing current.

Caudal Nerve Action Potentials were recorded with the active recordingelectrode positioned 10 mm below the hair line on the tail (determinedvisually) and the stimulating cathode 60-70 mm further distal. Velocitywas calculated by dividing the distance between the stimulating cathodeand the active electrode by the absolute onset latency of the initialdepolarizing current.

Tibial Motor Conduction was recorded with the active electrodepositioned in the intrinsic muscles of the hindpaw and the stimulatingcathode proximal to the ankle, behind the lateral malleolus.

Preparation of Animals: During all recording sessions, animals wereanesthetized with isoflurane and placed in a prone position. Respirationand temperature was monitored during the electrophysiologic recordingprocedure.

Electrodes: The placement of the active, reference and ground electrodeswas tailored to each modality and positioned with respect to bonylandmarks in each subject. Platinum needle electrodes (Grass-Telefactor,Co.), with impedances of approximately 50 kohms @ 1,000 Hz, were used asboth active and reference leads for all PNS recordings.

Temperature Control: Rectal temperature was maintained between 35.5 and38.0 degrees C. throughout the recording sessions.

Data Processing: Neuroelectric signals were impedance matched usingunity gain preamplifiers, appropriately band-passed using multi-polefilters, and further differentially amplified using a gain factor of0.5-50K. The filter settings were adjusted for each modality. Commonmode rejection levels and gain factors were adjusted to minimize 60 Hzinterference and to optimize the signal-to-noise ratio for eachrecording series. The amplified signal was time-locked to the evokingstimulus, multiplexed into selected channels and digitized at a rategreater than 5 times the highest frequency sampled. The data was scannedfor artifacts (using a predetermined rejection level—80% of thedigitized window) and digitally averaged for an epoch appropriate forthe modality under study. The number of sweeps included in each averagewas adjusted for each measure to optimize the signal-to-noise ratio andfacilitate the accurate assessment of both onset latency and peakamplitude measures.

Scoring of Data: All electrophysiologic data was scored followingoptimization of the signal. Onset latency was measured from the stimulusartifact to the initiation of the depolarization to the nearest 0.01msec; amplitude was measured from baseline to the peak of thedepolarization to the nearest 0.01 μV for sensory responses, and to thenearest 0.01 mV for motor responses. All measurements were conductedwith an internal computer cursor that follows the digitized trace. Allwave forms were stored digitally and were available for further off-lineanalysis.

Calibration: The amplifiers and filters were calibrated onsite on eachday of electrophysiologic recordings.

Terminal Phase study conduct: Animals were anesthetized by CO₂inhalation followed by cardiac puncture at specific time points.

Results: Baseline measurements of Nerve Conduction Velocity (NCV)

The first NCV assessment occurred 8-9 days after the administration ofSTZ and after the presence of hyperglycemia was confirmed in each of therats in Groups 2-4 (see Table E4 above). At that time point, which wasprior to any administration of vehicle or PEGylated human C-peptide(ie., Baseline), there was clear and significant evidence of aSTZ-induced peripheral polyneuropathy. At the Baseline assessment,maximal caudal NCV was reduced by slightly more than 10 m/sec(approximately 18%) in each of the STZ-treated groups compared tofindings in the age-matched control group (FIG. 16A). At that timepoint, maximal digital NCV was reduced by 3-4 m/sec (FIG. 17A) andtibial distal latency was prolonged (consistent with slowed velocity) byapproximately the same 10% value. Week 4 measurements of NerveConduction Velocity (NCV)

FIGS. 16B and 17B illustrate the caudal and digital NCV in each of thefour groups after a 4-week period (from Baseline) of administration ofeither vehicle alone or PEGylated human C-peptide at either 1.0 or 3.0mg/kg/week. During this 4-week period, one rat in Group 2, five rats inGroup 3 and one rat in Group 4 died. The Baseline values in FIGS. 16Aand 17A have omitted data from these missing rats to keep thecomparisons across the same subset of subjects.

NCV in the control group remained relatively constant for both thecaudal and digital nerves over the examined 4-week period (FIG. 18).However, as expected, velocity in both the caudal and digital nervecontinued to decrease in the STZ-only group. For the purely sensorydistal digital nerve, there was an additional slowing of approximately 3m/sec (10%) over the initial 4-week treatment period (FIG. 18). Thecontinued slowing of NCV is consistent with progressive damage to thedistal nerves due to the STZ-induced destruction of pancreatic betacells, leading to hyperglycemia and endogenous C-peptide deficiency,which will eventually lead to altered transmembrane currents, changes inthe micro-environment at the nodes, axonal atrophy, and ultimately toWallerian degeneration.

The absolute latency of the tibial motor response was slightly longer atthe Week 4 assessment in all groups, reflecting continued animal growth,however there was little or no difference across groups from thismeasure over the course of the initial four weeks of the study (data notshown).

NCV decreased at a slower rate compared to the findings in the STZ onlygroup for the survivors in each of the groups co-treated with PEGylatedhuman C-peptide over the 4-week period from Baseline (FIG. 18). Howeverat the 4-week time point the effects were small.

Week 8 Measurements of Nerve Conduction Velocity (NCV)

FIGS. 16C and 17C illustrate the caudal and digital NCV in each of thefour groups at the 8 week time point. Over the study period, NCV in thecontrol group for the mixed caudal nerve increased by approximately 4m/sec (7%) from Baseline values (FIG. 19). This change is consistentwith the well documented continued post-natal increases in myelin andaxonal cross-sectional diameter. No additional animals in any group diedbetween the 4^(th) and 8^(th) week of the post Baseline assessment.

There was relatively little change in the NCV for the digital nerve inthe control group over the 8-week period from baseline (FIGS. 18 and19). In contrast, NCV in the STZ only group (Group 2) continued todecline over this period. This progressive deterioration is consistentwith the continued insult induced by hyperglycemia and lack ofendogenous C-peptide production. By Week 8, digital NCV in the STZ-onlygroup was decreased by >4 m/sec (14%) from Baseline and by more than 20%from values in the age-matched control group. In contrast, digital nerveNCV was either stable (Group 3) or actually improved (Group 4) over the8 week study period in the groups co-treated with PEGylated humanC-peptide (FIG. 18).

Tables E11 and E12 outline the percent change in the digital and caudalNCV, respectively from the Baseline to the 8-week assessment time point.

TABLE E11 Percent change in the digital NCV Digital Nerve NCV (m/sec)Change from Baseline to Baseline Week 4 Week 8 Week 8 Control (No STZ)34.4 34.0 32.9 −4.4% Vehicle Control(No 30.5 27.3 26.2 −14.1% PEGylatedC- peptide) PEGylated human C- 29.7 28.8 29.8 +0.3% peptide (1 mg/kg/week) PEGylated human C- 28.6 27.8 30.1 +5.2% peptide (3 mg/kg/ week)

TABLE E12 Percent change in the caudal NCV Caudal Nerve NCV (m/sec)Change from Baseline to Baseline Week 4 Week 8 Week 8 Control (No STZ)53.0 52.9 56.8 +7.2 Vehicle Control (No 41.1 40.3 42.1 +2.4 PEGylatedC-peptide) PEGylated human C- 42.1 43.2 46.1 +9.5 peptide (1 mg/kg/week) PEGylated human C- 43.8 44.3 46.1 +5.3 peptide(3 mg/kg/ week)

Conclusions: There was a substantial slowing of both the digital andcaudal NCV in the groups treated with STZ (Groups 2-4) compared to theage-matched control group (Group 1). These effects were evident atBaseline, 8-9 days after the administration of the STZ, but prior to theco-administration of PEGylated C-peptide.

A progressive slowing of NCV was documented for the digital nerve overthe 8 week study period in the group treated with STZ only. Theco-administration of PEGylated C-peptide, at either 1.0 or 3.0mg/kg/week, prevented this continued deterioration. In the groupco-treated with 3.0 mg/kg/week of PEGylated C-peptide there was evenslight improvement of digital nerve NCV in the 8 week period followingan STZ-induced neuropathy.

The results from this study clearly suggest that over the time periodexamined, the co-treatment with PEGylated C-peptide providedneuroprotection against the neuropathy induced by STZ alone. This effectwas especially evident for the purely sensory digital nerve. Due in partto the early loss of subjects, this study provides only initial insightsinto dose-related different in the benefits of PEGylated C-peptide, butthere is a suggestion in the digital data supporting slightly greaterbenefits for the high dose group.

The caudal nerve data demonstrated a substantial negative impact of theSTZ treatment which was manifest at Baseline in Groups 2-4. There was nofurther evidence of slowing in caudal NCV during the 8 week studyperiod. However, there was improvement in velocity for the two groupsco-treated with PEGylated C-peptide that approximated the trend in thecontrol group (Table E12). The improvement in Groups 3 and 4 weregreater than that observed in the STZ only group. As was the case forWeek 4 there is little change in the tibial motor responses acrossgroups (data not shown).

These results demonstrate that the biological activity of the nativeC-peptide is retained when the peptide is PEGylated, which extends itscirculating half-life and thereby lessens the frequency of replacementdosing. The average maximum plasma concentration assessed 2 days afterdosing in the third week in the low-dose, and high-dose PEGylated humanC-peptide, and PEGylated rat C-peptide groups was approximately 129 nM,431 nM, and 12 nM, respectively (data not shown). The average minimumplasma concentration at the end of the study was approximately 22 nM, 94nM, and 2 nM in the low-dose and high-dose PEGylated human C-peptide,and PEGylated rat C-peptide groups, respectively. It is concluded thatPEGylated human C-peptide retains the beneficial biological propertiesof the unmodified C-peptide, and is effective for both the treatment andprevention of the long complications of diabetes, In particular thecurrent experiments establish that human PEGylated C-peptide iseffective for the treatment of neuropathies associated with diabetes.

Example 12 GMP Batch Preparation of Pegylated C-Peptide Overview

PEGylation: The synthesis of the human PEGylated C-peptide was carriedout in a single step by coupling of the N-terminus of the humanC-peptide (sodium salt) with the branched, approx. 40 kDa-NHS ester PEGderivative (SUNBRIGHT GL2-400GS2 (NOF Corporation) in the presence ofN-methyl morpholine.

SUNBRIGHT GL2-400GS2 (115 g) is first dissolved in 600 mL of a solutionof (50/50) acetonitrile/water. The resulting solution was stirred andcharged with another solution containing human C-peptide (7.9 g) in asolution of 175 mL of acetonitrile/water, followed by addition of 1.2 mLof N-Methyl Morpholine (NMM). Addition of NMM was repeated several timesat −1 hr intervals, with the progress of the reaction monitored by HPLCprior to each addition. This process was repeated about 8 to 10 timesand then the reaction was stirred overnight for about 8 to 12 hrs. Theresulting reaction mixture was carried on to the purification step oncethe reaction was verified as complete by HPLC analysis. Typically duringthis process several sub-lots were prepared and then combined forpurification as described below.

Purification of Crude PEGylated C-Peptide by Preparative Reversed PhaseChromatography

The crude PEGylated C-peptide solution was diluted with 6 volumes in0.1% TFA/water. The pH was adjusted to a pH of ≦3 and purified bypreparative HPLC using reverse phase silica (Diasogel C-18, 15 μm, 300Angstrom). The adsorbed PEGylated C-peptide was eluted from the columnby applying a gradient of acetonitrile in dilute aqueous TFA (Buffer Ais 0.1% TFA, Buffer B is 100% ACN: 0 to 25% B in 5 minutes, then 25% to50% B during 100 minutes and then hold until the product is eluted). Theeluate was monitored by UV at 230 nm. Fraction with purity of ≧90,NSI >6.0% are pooled. Fractions with purity >70% maybe recycled.

Desalting and Purification of PEGylated C-Peptide by PreparativeReversed Phase Chromatography

The combined pure fractions obtained from the preceding step weredesalted and purified by preparative HPLC using reverse phase silica.The column was washed with dilute aqueous TFA, followed by diluteaqueous ammonium acetate. The PEGylated C-peptide was then eluted fromthe column by applying a gradient of acetonitrile in dilute aqueous AcOH(Buffer A is 2% acetic acid, Buffer B is 100% ACN: 0 to 25% B in 5minutes, then 25% to 50% B during 75 minutes and then hold until theproduct is eluted). The eluate was monitored by UV at 230 nm. The purefractions obtained from chromatography were pooled (purity ≧95%,NSI >3.0%) and lyophilized. Fractions with purity >80% maybe recycledfor further purification.

Ion Exchange Purification of PEGylated C-Peptide by Preparative HPLC

The crude lyophilized PEGylated Human C-peptide from the step above(˜180 g) was dissolved in 5% acetonitrile/water and applied to an ionexchange column (DEAE52 Cellulose). The column was then washed with ˜50L of water and the product was eluted off the column with ˜40 L of anaqueous solution of sodium chloride (1M)/ammonium acetate (1M). Theeluate was monitored by UV at 230 nm. The pure fractions obtained fromthe chromatography were pooled (≧92% purity; no single impurity(NSI) >4%) and carried on for desalting/purification. Fractions withpurity >80% maybe recycled.

Desalting and Purification of CBX129801 by Preparative Reversed PhaseChromatography

The pure fractions from the ion exchange chromatography step werediluted with an equal volume of water and applied to a preparative HPLCcolumn (silica). The column was then washed with dilute 2% acetic acid(1 BV) and the product eluted with a solution of acetonitrile in diluteacetic acid (Buffer A is 2% acetic acid, Buffer B is 100% ACN: 0 to 25%B in 5 minutes, then 25% to 50% B during 50 minutes and then hold untilthe product is eluted). The eluate was monitored by UV at 230 nm. Thepure fractions (purity ≧95%, NSI >3.0%) obtained from chromatographywere pooled and lyophilized. Fractions with purity >80% maybe recycled.

Lyophilization of PEGylated C-Peptide

The product from the preceding purification was reconstituted at aconcentration of about 15-20 g/L in 2% aqueous acetic acid andlyophilized to give the pure PEGylated C-peptide drug substance as itsfree acid.

Example 13 Biophysical Characterization of PEGylated C-Peptide

A batch of the PEGylated C-peptide prepared as described in Example 12above, with purity of 99.5%, as determined by RP-HPLC with UV detection,was used in the analytical investigations described below unless notedotherwise. The structural studies conducted are listed in Table E13. Allanalyses confirm the chemical structure of the drug substance.

TABLE E13 Structural testing performed Test Analytical TechniqueMolecular mass MALDI-TOF MS Identity FT-IR Identity and ratios ofindividual Amino acid analysis for DS amino acids Identity and chiralityof individual Chiral amino acid analysis amino acids Molecular mass andsequence of amino CID-MS/MS acids (performed at the FI stage) PeptideMapping (to confirm sequence Chymotrysin digest followed on PEGylatedpeptide) by HPLC and MS/MS analysis of fragments Absence of Counter ionIon chromatography, RP-HPLC, ICP-MS

In addition to the structure elucidation tests, described above,additional characterization studies were performed on the PEGylatedC-peptide described in Example 12 above, and these additional studiesare listed in Table E14.

Molecular mass by MS: Matrix Assisted Laser Desorption Ionization-Timeof Flight (MALDI-TOF) was used to verify the molecular mass of the drugsubstance. The sample gave a positive ion MALDI-TOF mass spectrum with abroad singly-charged pseudomolecular ion cluster observed centeredapproximately at m/z 45743.

Fourier Transform Infrared Spectroscopy (FT-IR): FT-IR spectra ofC-peptide, the PEG reagent, and PEGylated C-peptide were collected on aJasco 4200 FT-IR spectrometer equipped with a TGS detector and asingle-bounce ZnSe crystal mounted on a ATR accessory. Solid sampleswere pressed against the Zn Se crystal with a Teflon rod. Residualmoisture peaks were subtracted from the spectra. The results are shownin FIG. 20 and FIG. 21 (expanded region). The spectrum of PEGylatedC-peptide is very similar to the spectra of the PEG reagent. This is notsurprising given the mass ratio of peptide to PEG.

However, there is a slight difference in the amide I region as shown inFIG. 21. Specifically, the amide I bands of PEGylated C-peptide show twopeaks at 1627 and 1653 cm⁻¹, while the spectrum of free C-peptide onlyexhibits one broad amide I band peak at 1634 cm⁻¹. An amide I band near1630 cm⁻¹ is normally associated with β-sheet structures or β-turns,while an amide I band near 1650 cm⁻¹ is normally assigned to α-helix,irregular, or random coil structures. The appearance of an absorbance at1653 cm⁻¹ is consistent with a more random structure for the PEGylatedpeptide compared to C-peptide.

To investigate if the difference in the amide I region is due todifferences in hydrogen bonding between amide groups and solvent water,the FT-IR spectra were collected in D₂O as shown in FIG. 22 and FIG. 23.For the collection of D₂O spectra, sample in D₂O solution was placedbetween two CaF₂ windows with a 6 μm spacer.

The FT-IR spectrum of PEGylated C-peptide in D₂O shows minimum amide IIband intensity (at 1566 cm⁻¹), which indicates all amide groups undergoH-D exchange. Upon H-D exchange, the amide II band is shifted from 1566to 1465 cm⁻¹ (becomes an amide II′ band). There is remaining amide IIintensity for free C-peptide at both higher (˜25 mg/mL) and lowerconcentrations (˜12.5 mg/mL) in D₂O, which indicates some un-exchangedamide groups. The un-exchanged amide groups are likely protected byeither intra-molecular hydrogen bonds within beta-turns orinter-molecular hydrogen bonds formed among peptide oligomers(aggregates) at high concentration. For the PEGylated C-peptide in D₂O,the effective C-peptide concentration is much lower because of the lowmass ratio of C-peptide to the 40 kDa PEG.

However, as can be seen from the second derivative FT-IR spectra (shownin FIG. 23), the amide I′ band for the high concentration sample ofC-peptide (˜25 mg/mL) shows a major peak at 1639 cm⁻¹, with a shoulderat 1645 cm⁻¹, whereas the low concentration sample (˜12.5 mg/mL) showsmajor peaks at both 1639 cm⁻¹ and 1645 cm⁻¹. This indicates thedifference in the amide I′ region may be concentration related. Incomparison to the spectrum of PEGylated C-peptide, the spectra of freeC-peptide shows more intensity near 1635-1640 cm⁻¹, indicating moreβ-turn structures in free C-peptide. It should be noted that signal tonoise was poor for more dilute samples of C-peptide samples precludingassessment of lower concentrations.

Identity and Ratio of Individual Amino Acids by Amino Acid Analysis: Toensure the identity and the correct ratio of the constituent aminoacids, amino acid analysis was performed on the PEGylated C-peptideprepared in Example 12. This method involves hydrolyzing the peptide instrong acid, separating the amino acids on an ion-exchange column, and,finally, detecting the eluents after ninhydrin derivatization. Theresults of the study are shown in Table E14. The results from the aminoacid analysis confirm the identity and theoretical relative occurrenceof amino acids in the PEGylated C-peptide within experimental error.

TABLE E14 Results of amino acid analysis Theoretical Observed AminoRelative Relative Acid Occurrence Occurrence Asp 1 1.1 Pro 2 2.1 Ser 22.2 Glx* 8 6.9 Gly 7 7.3 Ala 3 3.0 Val 2 2.0 Leu 6 6.5 Notes: *Glx =results from Gln + Glu.

Identity and Chirality of Individual Amino Acids by GC: Chiral aminoacid analysis of Example 12 was performed to investigate the chiralidentity of the constituent amino acid residues. The peptide ishydrolyzed in deuterated solvents (DCI/D₂O), derivatized as theN(O,S)-fluoroacetyl amino acid esters, and analyzed with GC-MS todetermine each amino acid enantiomer. GC was performed using adeactivated glass capillary coated with Chirasil-Val. The carrier gaswas hydrogen. The results are shown in Table E15. The values obtainedconfirm the chirality expected for the amino acids constituting thestructure of the PEGylated C-peptide of Example 12.

TABLE E15 Results of chiral amino acid analysis Amino Acid Content ofL-amino Acid (%) Asp >99.9 Pro 99.86 Ser 99.51 Glx >99.9 Ala 99.9Val >99.9 Leu 99.89

Sequence of amino acids by MS/MS: Given the large size andpolydispersity of the PEG, sequencing by MS/MS is performed at the FinalIntermediate stage. The amino acid sequence of the PEGylated C-peptideof Example 12 was investigated by performing MS/MS using CID (CollisionInduced Dissociation), a technique in which the intact sample moleculeis deliberately fragmented with the intention of gaining structuralinformation from the product ion spectrum created by the process.

The types of fragment ions observed in a MS/MS spectrum depend on manyfactors including primary sequence, the amount of internal energy, howthe energy was introduced, charge state, etc. The accepted nomenclaturefor fragment ions was first proposed by Roepstorff and Fohlman[Biomedical Spectrometry, 1984, 11(11): 601], and subsequently modifiedby Johnson et al. [Annals of Chemistry, 1987, 59(21): 2621-2625].

Fragments will only be detected if they carry at least one charge. Ifthis charge is retained on the N-terminal fragment, the ion is classedas either a, b, or c. If the charge is retained on the C-terminal, theion type is either x, y, or z. A subscript indicates the number ofresidues in the fragment.

In addition to the proton(s) carrying the charge, c ions and y ionsabstract an additional proton from the precursor peptide. Thus, sixsingly-charged sequence ion are possible. Note that these structuresinclude a single charge-carrying proton. In electrospray ionization,peptides generally carry two or more charges, so that fragment ions maycarry more than one proton.

The expected, multiply-charged b and y and fragment ions were calculatedusing a computer program developed by Croker et al. [Journal ofBiomolecular Techniques, 2000, volume 11, issue 3, 135-141]. The resultsare shown in Tables E16 and E17. Fragmentation and sequence analysis byMS/MS and MS/MS/MS confirmed the suggested primary sequence of thePEGylated C-peptide of Example 12 final intermediate.

TABLE E16 Summary of MS Fragmentation and sequence analysis N-terminalIon Series Sequence Expected Observed Expected Observed ExpectedObserved Example 12 Pos. b¹⁺ m/z b²⁺ m/z b³⁺ m/z Glu b1 130.1 — 65.5 —44 — Ala b2 201.1 201.1 101.1 — 67.7 — Glu b3 330.1 330.1 165.6 — 110.7— Asp b4 445.2 445.1 223.1 — 149.1 — Leu b5 558.2 558.2 279.6 — 186.8 —Gln b6 686.3 686.2 343.7 — 229.4 — Val b7 785.4 785.3 393.2 — 262.5 —Gly b8 842.4 842.3 421.7 — 281.5 — Gln b9 970.5 970.4 485.7 — 324.2 —Val b10 1069.5 1069.5 535.3 — 357.2 — Glu b11 1198.6 1198.4 599.8 —400.19 — Leu b12 1311.6 1311.6 656.3 — 437.9 — Gly b13 1368.7 1368.6684.8 684.8 456.9 — Gly b14 1425.7 1425.6 713.4 713.3 475.9 — Gly b151482.7 1482.6 741.9 741.8 494.9 — Pro b16 1579.8 790.4 — 527.3 — Gly b171636.8 1636.8 818.9 818.8 546.3 — Ala b18 1707.8 1707.8 854.4 854.3569.9 — Gly b19 1764.8 1764.9 882.9 882.8 589 — Ser b20 1851.9 1851.8926.4 926.4 618 — Leu b21 1965 1964.9 983 982.9 655.7 — Gln b22 2093 —1047 1046.9 698.3 — Pro b23 2190.1 — 1095.5 — 730.7 — Leu b24 2303.2 —1152.1 1151 768.4 — Ala b25 2374.2 — 1187.6 1187.5 792.1 — Leu b262487.3 — 1244.1 1244.1 829.8 — Glu b27 2616.3 — 1308.7 1308.6 872.8 —Gly b28 2673.3 — 1337.2 1337.1 891.8 — Ser b29 2760.4 — 1380.7 1380.6920.8 — Leu b30 2873.5 — 1437.2 1437.1 958.5 958.3 Gln b31 3001.5 —1501.3 1501.2 1001.2 — OH — — — — — — —

TABLE E17 Summary of MS Fragmentation and sequence analysis C-terminalIon Series Sequence Expected Observed Expected Observed ExpectedObserved Example 12 Pos. y¹⁺ m/z y²⁺ m/z y³⁺ m/z Glu y31 3019.5 — 1510.31510.3 1007.2 1007.2 Ala y30 2890.5 — 1445.7 — 964.2 — Glu y29 2819.4 —1410.2 — 940.5 — Asp y28 2690.4 — 1345.7 1345.5 897.5 — Leu y27 2575.4 —1288.2 1288.1 859.1 — Gln y26 2462.3 — 1231.7 1231.5 821.4 — Val y252334.2 — 1167.6 1167.5 778.8 — Gly y24 2235.2 — 1118.1 1117.9 745.7 —Gln y23 2178.1 — 1089.6 1089.4 726.7 — Val y22 2050.1 — 1025.5 — 684 —Glu y21 1951 1951 976  975.9 651 — Leu y20 1822 1821.9 911.5  911.4 908— Gly y19 1708.9 1708.8 855 — 570.3 — Gly y18 1651.9 1651.9 826.4  826.4551.3 — Gly y17 1594.8 1594.9 797.9  797.8 532.3 — Pro y16 1537.8 1537.8769.4  769.3 513.3 — Gly y15 1440.8 — 720.9 — 480.9 — Ala y14 1383.81383.7 692.4 — 461.9 — Gly y13 1312.7 1312.6 656.9 — 438.2 — Ser y121255.7 1255.6 928.4 — 419.2 — Leu y11 1168.7 1168.6 584.8 — 390.2 — Glny10 1055.6 1055.5 528.3 — 352.5 — Pro y9 927.5 927.5 464.3 — 309.8 — Leuy8 830.5 — 415.7 — 277.5 — Ala y7 717.4 717.3 359.2 — 239.8 — Leu y6646.3 646.3 323.7 — 516.1 — Glu y5 533.3 533.2 267.1 — 178.4 — Gly y4404.2 404.2 202.6 — 135.4 — Ser y3 347.2 347.2 174.1 — 116.4 — Leu y2260.2 260.2 130.6 — 87.4 — Gln y1 147.1 — — 49.7 — OH — — — — — — —

Peptide Mapping: A peptide map is a fragmentation pattern generated bydigestion of a protein with proteolytic enzymes. The pattern of peptidefragments is characteristic of a particular protein and may be used toidentify structure. A method for mapping C-peptide was previouslydeveloped and four fragments were identified by mass spectrometry. Thefour fragments contain amino acids 25-31 (labeled as fragment A), 13-24(labeled as fragment B), 1-12 (labeled as fragment C), and 1-24 (labeledfragment as D) as shown on the bottom panel of FIG. 24.

A side-by-side comparison was performed wherein C-peptide (1 mg/mL) andPEGylated C-peptide (10 mg/mL) were dissolved in 25 mM ammoniumbicarbonate buffer. To each 1 mL of sample, 40 μl_of 0.25 mg/mLchymotrypsin was added and the samples were incubated for four hours at37° C. The digestion was stopped by the addition of formic acid, and thesamples were analyzed by RP-HPLC. The results are shown in FIG. 24.

As expected for PEGylated C-peptide, fragment C (1-12) and D (1-24) werenot observed since the PEG moiety is attached at the N-terminus.Fragments 25-31 and 13-24 were observed for the PEGylated C-peptide ofExample 12. To investigate whether the peak at 14 minutes was undigestedPEGylated C-peptide, a time course study for the digestion was performedover 27 hours. No additional fragments were obtained consistent with thedigestion going to completion. In addition, a 50/50 mixture ofundigested PEGylated C-peptide and digested PEGylated C-peptide wasanalyzed by RP-HPLC with an extended gradient to see if any separationcould be achieved; however, only a single peak was observed. Thereforeit is concluded that the peak at 14 minutes contains PEGylated 1-12 and1-24 fragments and possibly some intact PEGylated C-peptide of Example12. The inability to resolve these fragments is not unexpected since thechromatographic behavior of the molecule is dominated by the large PEGmoiety.

Absence of Counterion: The ammonium content was measured by IonChromatography (IC), acetic acid by HPLC, and sodium content by ICP/MSto assure little or no counter ion remained after the desaltingprocedure.

The ammonium content was determined to be 0.035% w/w, and the sodiumcontent was found to be 0.02% w/w, below the specification limit.

Although the levels of counterions in the drug substance were low, whencalculated on a molar basis, may be indicative of some association ofammonia (0.9 molar ratio) and or sodium (0.4 molar ratio) to the finaldrug substance.

Sedimentation Velocity by Analytical Ultracentrifugation: To assess thehomogeneity and distribution of any aggregates in PEGylated C-peptide,the sedimentation velocity was measured in an analyticalultracentrifuge. Using this technique, aggregates can be detected on thebasis of their different sedimentation coefficients. Sedimentationvelocity is an absolute method based on simple physical principles. Itscalibration is based on fundamental units of length and time, requiringno standard molecules as reference. Sedimentation coefficients depend onmolecular shape as well as molecular mass, thus it is not possible topredict the sedimentation coefficient for an oligomer even when themonomer sedimentation coefficient is known.

The normalized sedimentation coefficient distribution for PEGylatedC-peptide lot 1008-134 (at ˜0.6 mg/mL) in PBS buffer is shown in FIG.25. The main peak at 0.802 S is 98.1%, indicating the sample ishomogenous. The sedimentation coefficient of C-peptide (unPEGylated) waspreviously determined to be in the range of ˜0.4-0.5 S, No signal inthis range was detected, indicating there is no free C-peptide. Inaddition, the sedimentation coefficient is consistent with a 40 kDabranched PEG (0.82 S).

Circular Dichroism Analysis of C-peptide and PEGylated C-peptide: Nearand far UV Circular Dichroisn (CD) analysis was performed on C-peptideand PEGylated C-peptide. Samples were dissolved in 20 mM phosphatebuffer containing 4.7% sorbitol, pH 6.0 at 1 mg/mL for C-peptide and˜10.4 mg/mL for PEGylated C-peptide (equivalent to 0.69 mg/mL ofC-peptide). The solvent subtracted spectrum was converted to the meanresidue ellipticity using the peptide concentration (1 or 0.69 mg/mL),the mean residue weight (97.4) and the path-length of the cell (1 cm forthe absorbance measurement or 0.02 cm for the CD). Measurements werecarried out on a Jasco J-715 spectropolarimeter.

As shown in FIG. 26, the mean residue ellipticity of C-peptide (upperline and PEGylated C-peptide (lower line) in the near UV region isessentially zero for both samples as there are no aromatic groups anddisulfide bonds (shown in the upper panel of FIG. 26).

The far UV CD spectra of C-peptide and PEGylated C-peptide show thesecondary structure is largely disordered. There is no double minima at220 and 208 nm typical for a α-helix and no valley at 217 nm typical foranti-parallel β-sheet.

CD analysis shows a nearly identical spectral shape for C-peptide andPEGylated C-peptide when corrected for concentration (lower panel ofFIG. 26) (note there is some error in the concentration estimates as thesample weights were not corrected for water or salts/solvents).Therefore, it can be concluded that PEGylation does not alter thesecondary structure of the peptide.

Size Exclusion Chromatography (SEC): A sample of the PEGylated C-peptideof Example 12 (100 μg in 20 mM phosphate buffer, 4.7% sorbitol, pH 6.0)was analyzed by size exclusion chromatography as shown in FIG. 27.

As part of the SEC method qualification, a 20 kDa PEGylated peptide wasindependently synthesized and analyzed by SEC to show the method wascapable of distinguishing related compounds based on size. An overlay ofthe chromatogram of the kDa PEGylated C-peptide (both samples at 100 μgload, in the same buffer system) with the PEGylated C-peptide of Example12 is shown in FIG. 28. As can be seen in FIG. 28, peaks of lowermolecular weight elute later from the SEC column. The absence of peaksbefore the main peak indicates there are no appreciable levels of highermolecular weight species present in the PEGylated C-peptide of Example12. Similarly, the absence of peaks after the main peak indicates thereare not appreciable levels of lower molecular weight species.

SDS-PAGE: Gel electrophoresis was conducted using a 4-12% Tris-Glycinegel. Molecular weight standards (see Blue Plus2, Prestained Standardsfrom Invitrogen) were applied in Lanes 2 and 10 as displayed in FIG. 29.Different amounts of PEGylated C-peptide ranging from 2 μg to 10 μg wereapplied to the gel in Lanes 4, 6, and 8. A single intense band between64-98 kDa was visualized by Coomassie staining. The hydrodynamic radiusof PEG is known to be greater than the size predicted based on themolecular weight of the protein markers. Therefore, this result is notunexpected. The SDS-PAGE results also show the absence of other highermolecular weight impurities.

Activity Profiling: Samples of PEGylated C-peptide, were compared toauthentic unlabeled C-peptide to confirm that the PEGylated productretained the activity of the unlabelled peptide.

Methods: Human Kidney (HK2) cells were seeded at a density of 20,000cells/well in (non-coated) 96 well (bl/cl) plates and incubated for 48hours. On the day of the experiment, HK2 cells were washed and starvedin DMEM+0.5% BSA for 1 hour. Cells were treated with 1 nM (finalconcentration) with ten replicates for 5 minutes. C-Peptide PEG GMP(lot#1-FIN-0988, C-Peptide PEG Tox (1007-119), C-Peptide PEG Tox(1008-090), unmodified C-Peptide (209400-3) and C-Peptide PEG reference(1008-134) were added in equal volumes. Plates were spun at 1000 rpm for5 minutes. The total treatment time was 7-10 minutes. Immediately aftertreatment, cells were fixed with 2% (final) paraformaldehyde andpermeabilized with ice-cold methanol. Cells were then treated withanti-pERK antibody and the plates were processed using the IF+Tyramideamplification, following standard protocols.

Results: The results shown in FIG. 30, demonstrate that the PEGylatedC-peptide retains the activity of the un-PEGylated product, and thisactivity is consistent across several different lots of C-peptide.

Example 14 Pharmaceutical Development

Since the PEGylated C-peptide (Example 1) is formulated as an aqueoussolution, key physicochemical properties that can affect the performanceof the formulation are solubility of the drug substance, pH, ionicstrength, and tonicity. All of these factors may impact the stability ofthe PEG portion of the PEGylated C-peptide and have been evaluated here.

At 20 mg/mL, the drug substance is well below the solubility limit inthe formulation buffer (up to 100 mg/mL). Excipients that are compatiblewith the drug substance have been selected to optimize the stability ofthe product as described below. The excipients used in the drug productare sodium phosphate (monobasic and dibasic), sorbitol, sodiumhydroxide, and distilled Water for Injection. All excipients arecompendial and meet the standards outlined in the USP. The choice ofexcipients and their levels are described in this example.

Buffer Selection: Phosphate buffer was selected for initial evaluationbecause it is commonly used in pharmaceutical preparations and has goodbuffering capacity at physiological pH. A pH screening study wasconducted by dissolving ˜1 mg/mL of PEGylated C-peptide (Example 12) in10 mM sodium phosphate buffer at varying pH (6.0, 6.5, 7.0, and 7.5).Samples were stored at 40° C. for 9 days and then analyzed by RP-HPLC.All samples were significantly degraded (˜40% or more), consistent withdegradation of the PEG component of the peptide-PEG conjugate. Inaddition, the pH of all formulations shifted downward by 0.7-1.0 pHunits indicating there was insufficient buffering capacity.Nevertheless, a clear trend could be observed with increased stabilityat lower pH. Namely the formulation starting at pH 6.0 was preferredover 6.5, 7.0, and 7.5, respectively. Therefore a target pH of 6.0 wasselected.

Tonicity Agent: A second formulation study was conducted to select atonicity agent (saline or sorbitol). The concentration of saline (0.9%)or sorbitol (4.7%) was selected to make the drug product solutionisotonic. The PEGylated C-peptide (Example 1) (1 mg/mL) was dissolved in20 mM sodium phosphate buffer with either saline (0.9%) or sorbitol(4.7%). The pH was adjusted to 6.0. Samples were stored at 5° C. and at40° C. The stability of these formulations was greatly improved by theaddition of a tonicity agent (data not shown). After 4 weeks at 40° C.,both tonicity agents gave equivalent results with a drop in area-%purity of ˜2-3%. After 12 weeks at 40° C., the sorbitol-containingformulation was clearly superior, with a drop in area-% purity of ˜7%compared to the saline-containing formulation which showed a drop inarea-% purity of ˜72% (corresponding to PEG degradation). At 5° C.,little to no degradation was observed in either formulation after 12weeks. Based on the accelerated stability results, sorbitol was selectedas the tonicity agent.

Ionic Strength: A third formulation study was conducted to evaluate theimpact of ionic strength on the stability of the formulation. Solutionsof PEGylated C-peptide (Example 12) were prepared at ˜20 mg/mL inphosphate buffer at 10, 20, and 50 mM containing 4.7% sorbitol andadjusted to pH 6.0. Samples were stored at 5° C. and 40° C. The resultsare summarized in Table E18.

TABLE E18 Stability of PEGylated C-peptide (Example 12) as a function ofbuffer concentration Initial 3 months at 5° C. 3 months at 40° C. BufferContent Purity Content Purity Content Purity Concentration (mg/mL) (%)pH (mg/mL) (%) pH (mg/mL) (%) pH 10 mM 22.3 99.4 5.9 21.4 99.3 6.0 8.140.3 4.1 20 mM 21.4 99.8 6.0 21.3 99.4 6.1 4.0 22.3 4.0 50 mM 22.1 99.96.1 21.1 99.6 6.2 4.2 21.8 4.9

At 5° C., all formulations looked similar after 3 months. There was noappreciable change in pH content or area-% purity. At 40° C., there wasa decrease in pH, purity, and content for all formulations. The bestresults were obtained for the 10 mM phosphate concentration, indicatinghigh ionic strength may negatively impact stability. Therefore, 10 mMphosphate was selected as the buffer concentration.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

1. A PEGylated C-peptide, wherein the PEGylated C-peptide has thestructure:

wherein R₁ is methyl, and n₁ and n₂ are within the range of about 400 to500, and the PEG moiety has a molecular weight of about 40 kDa.
 2. Apharmaceutical composition comprising the PEGylated C-peptide of claim 1and a pharmaceutically acceptable carrier or excipient.